Functional characterization of Nosema bombycis (microsporidia) trehalase 3.

Nosema bombycis, an obligate intracellular parasite, is a single-celled eukaryote known to infect various tissues of silkworms, leading to the manifestation of pebrine. Trehalase, a glycosidase responsible for catalyzing the hydrolysis of trehalose into two glucose molecules, assumes a crucial role in thermal stress tolerance, dehydration, desiccation stress, and asexual development. Despite its recognized importance in these processes, the specific role of trehalase in N. bombycis remains uncertain. This investigation focused on exploring the functions of trehalase 3 in N. bombycis (NbTre3). Immunofluorescence analysis of mature (dormant) spores indicated that NbTre3 primarily localizes to the spore membrane or spore wall, suggesting a potential involvement in spore germination. Reverse transcription-quantitative polymerase chain reaction results indicated that the transcriptional level of NbTre3 peaked at 6 h post N. bombycis infection, potentially contributing to energy storage for proliferation. Throughout the life cycle of N. bombycis within the host cell, NbTre3 was detected in sporoplasm during the proliferative stage rather than the sporulation stage. RNA interference experiments revealed a substantial decrease in the relative transcriptional level of NbTre3, accompanied by a certain reduction in the relative transcriptional level of Nb16S rRNA. These outcomes suggest that NbTre3 may play a role in the proliferation of N. bombycis. The application of the His pull-down technique identified 28 proteins interacting with NbTre3, predominantly originating from the host silkworm. This finding implies that NbTre3 may participate in the metabolism of the host cell, potentially utilizing the host cell's energy resources.

Effect of conventional and microwave heating treatment on antioxidant activity of quinoa protein after simulated gastrointestinal digestion.

Effects of microwave and traditional water bath treatment at different temperatures (70, 80, 90 ℃) on in vitro digestion rate and antioxidant activity of digestion products of quinoa protein were investigated. The results indicated microwave treatment at 70 ℃ produced the highest quinoa protein digestion rate and the strongest antioxidant activities of its digestion products (P < 0.05), which was further verified by the results of free amino, sulfhydryl group, gel electrophoresis, amino acid profiles and the molecular weight distribution of the digestion products. However, limited exposure of active groups induced by water bath treatment might decrease the susceptibility of digestive enzymes and subsequently lower the digestibility and antioxidant activities of quinoa protein. The results suggested that a moderate microwave treatment could be used as a potential way to enhance the in vitro digestion rate of quinoa protein, as well as increase the antioxidant activities of its digestion products.

Self-assembled biobased chitosan hybrid carrying N/P/B elements for polylactide with enhanced fire safety and mechanical properties.

The inherent shortcomings such as flammability, brittleness, and low crystallinity limit the broad applications of poly(lactic acid) (PLA). To improve the fire resistance and mechanical properties of PLA, a chitosan-based core-shell flame retardant additive (APBA@PA@CS) was prepared for PLA via the self-assembly of interionic interactions among chitosan (CS), phytic acid (PA), and 3-aminophenyl boronic acid (APBA). The peak heat release rate (pHRR) and total heat release rate (THR) of PLA composite containing 3 wt% APBA@PA@CS decreased from 460.1 kW/m2 and 75.8 MJ/m2 to 419.0 kW/m2 and 53.1 MJ/m2, respectively. The presence of APBA@PA@CS contributed to the formation of a high-quality char layer rich in phosphorus and boron in the condensed phase and released non-flammable gases in the gas phase to hinder the exchange of heat and O2, thereby having a synergistic flame retardant effect. Meanwhile, the tensile strength, elongation at break, impact strength, and crystallinity of PLA/APBA@PA@CS were increased by 3.7 %, 17.4 %, 5.3 %, and 55.2 %, respectively. This study provides a feasible route to construct a chitosan-based N/B/P tri-element hybrid to improve the fire safety performance and mechanical properties of PLA biocomposites.

Effect of compositional interaction on in vitro digestion of starch during the milling process of quinoa.

With the gradual stripping of cellulose and protein in the milling process, the proportion of starch gradually increased to 91.35 %. In particular, milling reduced the content of saponins from 5.06% to 0.23%. Milling increased the swelling power of quinoa flour but decreased the gelatinization enthalpy of quinoa starch. Accordingly, the microstructure of quinoa with higher milling degree after cooking was more uniform and denser. Fourier transform infrared (FTIR) analysis showed that starch-protein interaction was beneficial to protect the ordered structure of quinoa starch. The digestibility of protein and starch was interrelated, and the digestibility rate increased with the raising milling degree. By fitting the starch digestograms and combining the digestion kinetic model, three digestion patterns of quinoa starch during milling were determined. The results of the confocal laser scanning microscope showed that the physical barrier composed of protein matrix coated starch and starch-protein complex affected starch digestion.

Development of a Relative Similarity Degree Based Engineering Construction Multi-Attribute Decision Model and Its Application.

Generally, there are large amounts of uncertain factors in the multi-attribute decision system. By using the gray relational degree and fuzzy gray relational degree, the weights of the comprehensive indexes are extracted. Then, a novel decision model is established based on the concept of relative similarity degree. Finally, comparative research is carried out taking the maritime safety engineering construction in Hunan Province, China, as an example to verify that the developed model is rather effective and practical for its high resolution and sensitivity in multi-attribute decision.

Alkaloids from Corydalis decumbens modulate neuronal excitability.

Eight new alkaloids, including five isoquinoline alkaloids, a benzoazepine alkaloid, two isoindole alkaloids, and three synthetic alkaloids firstly obtained from the natural sources, together with three known ones were isolated from the bulbs of Corydalis decumbens. The structures were determined by analysis of their spectroscopic data and single-crystal X-ray diffraction. This is the first report of isoindole alkaloid and benzoazepine alkaloid from the genus Corydalis. Full NMR data for 9-11 are reported here for the first time. Moreover, the ability to modulate neuronal Ca2+ mobilization of the isolated alkaloids was tested in primary cultured neocortical neurons. Compound 7 inhibited spontaneous Ca2+ oscillations in primary neocortical neuron cultures at low micromolar concentrations.

New phthalideisoquinoline hemiacetal alkaloid derivatives from Corydalis decumbens.

Two new phthalideisoquinoline hemiacetal alkaloid derivatives, named corybensines A and B (1 and 2), and four known alkaloids (3-6) were isolated from the bulbs of Corydalis decumbens. Their structures were characterized by analysis of 1D/2D NMR and ECD data, quantum chemical ECD calculations, and X-ray diffraction analysis. Among them, compound 2 represents the first naturally occurring phthalideisoquinoline hemiacetal alkaloid derivative with a 2-pyrrolidinone moiety. The activity of the isolated compounds towards neuronal excitability was examined.

Phthalideisoquinoline Hemiacetal Alkaloids from Corydalis decumbens That Inhibit Spontaneous Calcium Oscillations, Including Alkyl Derivatives of (+)-Egenine That Are Strikingly Levorotatory.

The new phthalideisoquinoline hemiacetal alkaloids (2-7) and the known analogues (1 and 8) were isolated from the bulbs of Corydalis decumbens. The new compounds were characterized by analysis of their NMR spectroscopic data, chemical degradation syntheses, X-ray crystallography, and comparison of experimental and calculated ECD data. All the isolates were screened in vitro for inhibitory activity of spontaneous calcium oscillations in primary cultured neocortical neurons. Compounds 1-3 and 5-7 were found to be active in the suppression of spontaneous calcium oscillations with IC50 values of 6.8, 5.6, 11.6, 10.2, 8.3, and 3.1 µM, respectively. It was also observed that the presence of hydroxy, methoxy, and ethoxy groups at the remote stereogenic center C-7' of some isolated phthalideisoquinoline hemiacetal alkaloids could alter the preferred conformation and invert the sign of optical rotation, rather than this resulting from configurational isomerism at C-1 or C-9, and that the 3J1,9 coupling constants of these analogues varied accordingly. For example, compounds 1 and 6 are levorotatory, despite these molecules having the same carbon skeleton and absolute configuration as (+)-egenine. This emphasizes the potential risk of incorrectly assigning absolute configuration based only on observed coupling constants or optical rotation when comparing the data of new compounds with literature values for known analogues, especially within this class of molecules.

Alkaloids from Corydalis decumbens suppress neuronal excitability in primary cultures of mouse neocortical neurons.

Eight previously undescribed alkaloids, named corydemine, dihydrocorydemine, corydedine, 8,13-dioxo-14-hydroxytetrahydropalmatine, egenine-α-N-oxide, egenine-ß-N-oxide, 7'-O-ethylegenine-α-N-oxide, and 7'-O-ethylegenine-ß-N-oxide, together with three known ones, muramine, l-tetrahydropalmatine, and (+)-egenine, were isolated from the bulbs of Corydalis decumbens. Their structures were elucidated by comprehensive spectroscopic analysis and chemical correlation. The isolated compounds were tested for their ability to modulate neuronal excitability in primary cultured neocortical neurons. Four of the compounds, corydemine, dihydrocorydemine, muramine, and l-tetrahydropalmatine, inhibited neuronal excitability with IC50 values of 3.6, 16.7, 13.5 and 14.0 µM, respectively.

In vitro unfolding of insulin: characterization of intermediates and putative unfolding pathway.

The in vitro insulin unfolding had been studied using the "equilibrium unfolding" method where protein is unfolded by reducing reagents in the presence of trace amounts of oxidants such as oxidized glutathione. Nine intermediates were captured in the unfolding process, named as P1A, P2A, P3A, P4A, P3B, P4B, P5B, P6B, and P7B, which were all either A chain derivatives or B chain derivatives. No intermediate with inter-A-B chain disulfide was captured. Based on the character of the intermediates, their distribution during the unfolding process and the hypothetic "transient" intermediates, an in vitro putative unfolding pathway of insulin had been proposed. Besides, the comparison of the intermediates captured in unfolding with the intermediates captured in the refolding process of insulin revealed that both unfolding/refolding processes of insulin shared common intermediates. Based on these observations we suggested that the unfolding pathway of insulin was similar to the refolding pathway but flowed in the opposite direction.

Equilibrium folding of porcine insulin precursor in the presence of redox buffer: implications for the common intermediates shared by its unfolding/ refolding processes.

We use the procedure established for 'disulfide stability analysis in redox system' to investigate the unfolding process of porcine insulin precursor (PIP). Six major unfolding intermediates have been captured, in which four contain two disulfides, two contain one disulfide. Based on the characterization and analysis of the intermediates an unfolding pathway has been proposed, by which the native PIP unfolded through in turn 2SS and 1SS intermediates into fully reduced form. Besides, the comparison of the intermediates captured in PIP unfolding process with those intermediates captured in its refolding process revealed that some intermediates captured during both unfolding/refolding processes of PIP have identical disulfide pairing pattern, from which we suggest that the unfolding/refolding processes of PIP share some common intermediates but flow in the opposite direction.

The sequence determinant causing different folding behaviors of insulin and insulin-like growth factor-1.

Although insulin and insulin-like growth factor-1 (IGF-1) belong to the insulin superfamily and share highly homologous sequences, similar tertiary structure, and a common ancestor molecule, amphioxus insulin-like peptide, they have different folding behaviors: IGF-1 folds into two thermodynamically stable tertiary structures (native and swap forms), while insulin folds into one unique stable structure. To further understand which part of the sequence determines their different folding behavior, based on previous reports from the laboratory, two peptide models, [B9A][1-4]porcine insulin precursor (PIP) and [B10E][1-4]PIP, were constructed. The plasmids encoding the peptides were transformed into yeast cells for expression of the peptides; the results showed that the former peptide was expressed as single component, while the latter was expressed as a mixture of two components (isomer 1 and isomer 2). The expression results together with studies of circular dichoism, disulfide rearrangement, and refolding lead us to deduce that isomer 1 corresponds to the swap form and the isomer 2 corresponds to the native form. We further demonstrate that the sequence 1-4 plus B9 of IGF-1 B-domain can make PIP fold into two structures, while sequence 1-5 of insulin B-chain can make IGF-1 fold into one unique structure. In other words, it is the IGF-1 B-domain sequence that 1-4 allows IGF-1 folding into two thermodynamically stable tertiary structures; this sequence plus its residue B9E can change PIP folding behavior from folding into one unique structure to two thermodynamically stable structures, like that of IGF-1.