[Cloning and expression pattern analysis of abscisic acid receptor gene McPYL4 in Mentha canadensis].

Mentha canadensis is a traditional Chinese herb with great medicinal and economic value. Abscisic acid(ABA) receptor PYLs have important roles in plant growth and development and response to adversity. The M. canadensis McPYL4 gene was cloned, and its protein characteristics, gene expression, and protein interactions were analyzed, so as to provide genetic resources for genetic improvement and molecular design breeding for M. canadensis resistance. Therefore, the protein characteristics, subcellular localization, gene expression pattern, and protein interactions of McPYL4 were analyzed by bioinformatics analysis, transient expression of tobacco leaves, RT-qPCR, and yeast two-hybrid(Y2H) techniques. The results showed that the McPYL4 gene was 621 bp in length, encoding 206 amino acids, and its protein had the conserved structural domain of SRPBCC and was highly homologous with Salvia miltiorrhiza SmPYL4. McPYL4 protein was localized to the cell membrane and nucleus. The McPYL4 gene was expressed in all tissue of M. canadensis, with the highest expression in roots, followed by leaves, and it showed a pattern of up-regulation followed by down-regulation in leaves 1-8. In both leaves and roots, the McPYL4 gene responded to the exogenous hormones ABA, MeJA, and the treatments of drought, AlCl_3, NaCl, CdCl_2, and CuCl_2. Moreover, McPYL4 was up-regulated for expression in both leaves and roots under the MeJA treatment, as well as in leaves treated with AlCl_3 stress for 1 h, whereas McPYL4 showed a tendency to be down-regulated in both leaves and roots under other treatments. Protein interactions showed that McPYL4 interacted with AtABI proteins in an ABA-independent manner. This study demonstrated that McPYL4 responded to ABA, JA, and several abiotic stress treatments, and McPYL4 was involved in ABA signaling in M. canadensis and thus in the regulation of leaf development and various abiotic stresses in M. canadensis.

In Silico Functional and Structural Analysis of Non-synonymous Single Nucleotide Polymorphisms (nsSNPs) in Human Paired Box 4 Gene.

In human genome, members of Paired box (PAX) transcription factor family are highly sequence-specific DNA-binding proteins. Among PAX gene family members, PAX4 gene has significant role in growth, proliferation, differentiation, and insulin secretion of pancreatic ß-cells. Single nucleotide polymorphisms (SNPs) in PAX4 gene progress in the pathogenesis of various human diseases. Hence, the molecular mechanism of how these SNPs in PAX4 gene significantly progress diseases pathogenesis needs to be elucidated. For the reason, a series of bioinformatic analyzes were done to identify the SNPs of PAX4 gene that contribute in diseases pathogenesis. From the analyzes, 4145 SNPs (rsIDs) in PAX4 gene were obtained, where, 362 missense (8.73%), 169 synonymous (4.08%), and 2323 intron variants (56.04%). The rest SNPs were unspecified. Among the 362 missense variants, 118 nsSNPs were found as deleterious in SIFT analysis. Among those, 25 nsSNPs were most probably damaging and 23 were deleterious as observed in PolyPhen-2 and PROVEAN analyzes, respectively. Following all analyzes, 14 nsSNPs (rs149708455, rs115887120, rs147279315, rs35155575, rs370095957, rs373939873, rs145468905, rs121917718, rs2233580, rs3824004, rs372751660, rs369459316, rs375472849, rs372497946) were common and observed as deleterious, probably damaging, affective and diseases associated. Following structural analyzes, 11 nsSNPs guided proteins were found as most unstable and highly conserved. Among these, R20W, R39Q, R45Q, R60H, G65D, and A223D mutated proteins were highly harmful. Hence, the results from above-mentioned integrated comprehensive bioinformatic analyzes guide how different nsSNPs in PAX4 gene alter structural and functional characteristics of the protein that might progress diseases pathogenesis in human including type 2 diabetes.

Effects of Blanching Methods on Nutritional Properties and Physicochemical Characteristics of Hot-Air Dried Edible Insect Larvae.

Global meat consumption is increasing worldwide, however, supply remains lacking. Several alternative protein sources, such as cultured meat, plant-based protein production, and edible insects, have been proposed to overcome this shortage. Interestingly, edible insects are characterized by superior digestive and absorptive qualities that make them the ideal replacement for traditional protein production. This study aims to further the processing ability of insect protein by investigating the effects of various pre-treatment methods, such as blanching (HB), roasting (HR), and superheated steam (HS), on the nutritional properties and physicochemical characteristics of proteins extracted from Hermetia illucens larvae. The drying rate, pH value, color analysis, amino and fatty acid profile, as well as bulk density, shear force, and rehydration ratios of the above pre-treatment methods, were explored. HS was found to have the highest drying rate and pH value analysis showed that HB and HS samples have significantly higher values compared to the other modalities. Raw edible insects had the highest value in the sum of essential amino acid (EAA) and EAA index when compared to EAAs. HB and HS showed significantly lower bulk density results, and HS showed the highest shear force and the highest value in rehydration ratio, regardless of immersion time. Therefore, taking the above results together, it was found that blanching and superheated steam blanching pre-treatment were the most effective methods to improve the processing properties of H. illucens after hot-air drying.

Effects of alternating electric field assisted freezing-thawing-aging sequence on longissimus dorsi muscle microstructure and protein characteristics.

The current study investigates the influence of alternating electric field (AEF)-assisted freezing-thawing-aging sequence on the muscle microstructure and myofibrillar protein characteristics. Three treatments were used for longissimus dorsi (LD) muscle: only aging (OA), freezing-thawing-aging sequence (FA) and AEF-assisted freezing-thawing-aging sequence (EA). Compared with the FA and EA groups, the OA group showed considerably fewer cracks between muscle fibers and maintained the integrity of the Z-line as observed using scanning and transmission electron microscopy, respectively. Furthermore, the EA treatment effectively decreased myofibrillar fragmentation, myofibrillar protein aggregation, and protein oxidation, as shown by the myofibrillar fragmentation index, turbidity, and total sulfhydryl concentration. Analysis of surface hydrophobicity and the Fourier transform infrared, UV absorption, and fluorescence spectrums indicated that AEF minimized the alterations of protein secondary and tertiary structure alterations during aging after freezing.

Effects of Defatting Methods on the Physicochemical Properties of Proteins Extracted from Hermetiaillucens Larvae.

In this study, we investigated the effects of various defatting methods, including organic solvent (aqueous, acetone, ethanol, and hexane) extraction and physical (cold pressure) extraction, on the nutritional, physicochemical, and functional properties of proteins extracted from Hermetia illucens larvae. The total essential amino acid contents were higher with cold pressure protein extraction than other treatments. The surface hydrophobicity with cold pressure treatment was the lowest, and there were no significant differences among the other treatments. The protein solubility after defatting with organic solvent was higher than for other treatments. The nonreduced protein band at 50 kDa of the defatted protein prepared using organic solvent was fainter than in the cold pressure treatment. The cold pressure-defatted protein showed the highest emulsifying capacity, and the water extracted protein showed the lowest emulsifying capacity. Although organic solvents may be efficient for defatting proteins extracted from insects, organic solvents have detrimental effects on the human body. In addition, the organic solvent extraction method requires a considerable amount of time for lipid extraction. Based on our results, using cold pressure protein extraction on edible insect proteins is ecofriendly and economical due to the reduced degreasing time and its potential industrial applications.

Influence of Multifrequency Ultrasound-Assisted Freezing on the Flavour Attributes and Myofibrillar Protein Characteristics of Cultured Large Yellow Croaker (Larimichthys crocea).

The influence of multifrequency ultrasound-assisted freezing (UAF) as compared with single- and dual-UAF on the flavour, microstructure, and myofibrillar proteins (MPs) of cultured large yellow croaker was investigated to improve food quality in a sustainable way and address the major global challenges concerning food and nutrition security in the (near) future. Multifrequency UAF-treated samples had lower total volatile basic nitrogen values during freezing than single- and dual-UAF-treated samples. Thirty-six volatile compounds were identified by solid-phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) during freezing, and the multifrequency UAF-treated samples showed significant decreases in the relative contents of fishy flavoured compounds, including 1-penten-3-ol and 1-octen-3-ol. In addition, multifrequency UAF treatment better maintained a well-organised protein secondary structure by maintaining higher α-helical and ß-sheet contents and stabilising the tertiary structure. Scanning electron microscopy images indicated that the ice crystals developed by the multifrequency UAF were fine and uniformly distributed, resulting in less damage to the frozen large yellow croaker samples. Therefore, multifrequency UAF improved the flavour attributes and MP characteristics of the large yellow croaker samples. Overall, multifrequency UAF can serve as an efficient way for improving food quality and nutritional profile in a sustainable way.

Effects of organic solvent on functional properties of defatted proteins extracted from Protaetia brevitarsis larvae.

This study investigated the effect of aqueous fat separation and defatting using organic solvents (99% methanol, ethanol, and n-hexane) on the characteristics and functionality of proteins extracted from Protaetia brevitarsis. The defatting efficiency, amino acid composition, protein solubility, and technical properties were the highest when proteins were defatted using n-hexane. Proteins defatted using ethanol were similar in foam capacity and emulsifying capacity. Surface hydrophobicity decreased when using organic solvents, and excessive fat content disrupted the functional properties of the extracted proteins. Proteins extracted using the different solvents displayed different pH values. The pH of the aqueous extract was the lowest. CIE L* a* b* color values also differed using the different extraction methods. Although n-hexane might be the most efficient solvent for defatting the proteins extracted from edible insects, ethanol could also be used to obtain similar foam and emulsifying capacities.

Functional Properties of Extracted Protein from Edible Insect Larvae and Their Interaction with Transglutaminase.

Global concern about food supply shortage has increased interest on novel food sources. Among them, edible insects have been studied as a potential major food source. This study aimed to improve the functional properties of protein solutions extracted from Protaetia brevitarsis (PB) by use of transglutaminase (TG) as a cross-linking agent. After various incubation times (10, 20, 30, 60, and 90 min) with TG, the protein solutions were assessed with regard to their amino acid composition, protein nutritional quality, pH, color (yellowness), molecular weight distribution, thermal stability, foam ability (capacity and stability), and emulsion ability (capacity and stability). Incubation with TG changed the amino acid composition of the proteins and shifted the molecular weight distribution towards higher values, while improving the rest of the aforementioned properties. Since the incubation time for 90 min decreased the protein functionality, the optimum incubation time for cross-linking PB-derived protein with TG is 60 min. The application of TG to edible insect proteins ultimately increases its functionality and allows for the development of novel insect processing technology.

Machine learning-driven protein engineering: a case study in computational drug discovery.

Research and development in drug discovery will need to find significant efficiency gains if the industry is to continue generating novel drugs. There is great expectation for machine learning (ML) to provide this boost in R&D productivity, but to harness the full potential of ML, the generation of new, high-quality datasets will be necessary. Here, the authors present a platform that combines high-throughput display and selection data generation with ML. More specifically, deep learning is used to inform the directed evolution of novel biotherapeutics using DNA library synthesis, ultra-high throughput selections, and next generation sequencing. By combining the learnings of multiple in silico models, their platform enables multi-parameter optimisation across multiple important protein characteristics. They also present a model for benchmarking these ML-driven drug discovery platforms according to the accuracy of their underlying in silico models, in conjunction with the throughput of their empirical experimentation.

Genome-wide analysis of the MATE gene family in potato.

The multidrug and toxic compound extrusion (MATE) protein family is a newly discovered family of secondary transporters that extrude metabolic waste and a variety of antibiotics out of the cell using an electrochemical gradient of H+ or Na+ across the membrane. The main function of MATE gene family is to participate in the process of plant detoxification and morphogenesis. The genome-wide analysis of the MATE genes in potato genome was conducted. At least 48 genes were initially identified and classified into six subfamilies. The chromosomal localization of MATE gene family showed that they could be distributed on 11 chromosomes except chromosome 9. The number of amino acids is 145-616, the molecular weight of proteins is 15.96-66.13 KD, the isoelectric point is 4.97-9.17, and they were located on the endoplasmic reticulum with having 4-13 transmembrane segments. They contain only two parts of the exons and UTR without introns. Some members of the first subfamily of potato MATE gene family are clustered with At2g04070 and they may be related to the transport of toxic compounds such as alkaloids and heavy metal. The function of the members of the second subfamily may be similar to that of At3g23560, which is related to tetramethylammonium transport. Some members of the third subfamily are clustered with At3g59030 and they may be involved in the transport of flavonoids. The fifth subfamily may be related to the transport of iron ions. The function of the sixth subfamily may be similar to that of At4g39030, which is related to salicylic acid transport. There are three kinds of conserved motifs in potato MATE genes, including the motif 1, motif 2, and motif 3. Each motif has 50 amino acids. The number of each motif is different in the gene sequence, of which 45 MATE genes contain at least a motif, but there is no motif in ST0015301, ST0045283, and ST0082336. These results provide a reference for further research on the function of potato MATE genes.

Interactions between soy protein hydrolyzates and wheat proteins in noodle making dough.

Soy protein hydrolyzate has been used as supplements in wheat flour to enhance the nutritional value of its products, but it may negatively affect the gluten properties simultaneously. In order to explore the mechanism of this effect, protein characteristics including disulfide bond, protein composition, intermolecular force of dough proteins, and atomic force microscope images of gluten were obtained. Results showed that disulfide bonds in dough increased when soy protein hydrolyzate was added, but glutenin macropolymer decreased. Atomic force microscope images showed that gluten were weakened by soy protein hydrolyzate. Based on these results, a model was developed to describe the interaction between soy protein hydrolyzates and wheat proteins: soy protein hydrolyzates linked with wheat proteins through disulfide bond, disrupted the glutenins polymerization, thus hindered gluten networks formation. The interaction between wheat proteins and soy protein hydrolyzates in noodle making dough could be described with this model reasonably.