Functional Characterization Research Articles

Comparative transcriptomics analysis reveals stage-specific gene expression profiles associated with gamete formation in Allium sativum L.

Commercial cultivars of garlic, a popular condiment, are sterile, making genetic variation and germplasm innovation of this plant challenging. Understanding mechanism of gamete sterility in garlic and their key regulatory networks is therefore important for fertility restoration. In this work, we conducted a detailed phenotypic analysis of fertile and sterile garlic genotypes and found that enlargement of topset in the inflorescence of sterile genotypes led to abnormal flowers. Additional cytological observations showed that aberrant meiotic cytokinesis in sterile garlic ultimately resulted in pollen degeneration. Transcriptomics analysis of sterile and fertile genotypes identified possible molecular mechanisms underlying gamete abortion. A total of 100 710 differentially expressed genes (DEGs) between the fertile and sterile garlic flowers at three stages of gamete development were identified, many of which were involved in homologous chromosome synapsis during meiosis, MYB transcription factor regulation, ribosome biogenesis and plant hormone signal transduction. Taken together, these results provide insight into the molecular mechanisms and regulatory networks underlying gamete development in garlic and point to a set of candidate genes for further functional characterization.

Deep Learning-Based Self-Adaptive Evolution of Enzymes

AbstractBiocatalysis has been widely used to prepare drug leads and intermediates. Enzymatic synthesis has advantages, mainly in terms of strict chirality and regional selectivity compared with chemical methods. However, the enzymatic properties of wild-type enzymes may or may not meet the requirements for biopharmaceutical applications. Therefore, protein engineering is required to improve their catalytic activities. Thanks to advances in algorithmic models and the accumulation of immense biological data, artificial intelligence can provide novel approaches for the functional evolution of enzymes. Deep learning has the advantage of learning functions that can predict the properties of previously unknown protein sequences. Deep learning-based computational algorithms can intelligently navigate the sequence space and reduce the screening burden during evolution. Thus, intelligent computational design combined with laboratory evolution is a powerful and potentially versatile strategy for developing enzymes with novel functions. Herein, we introduce and summarize deep-learning-assisted enzyme functional adaptive evolution strategies based on recent studies on the application of deep learning in enzyme design and evolution. Altogether, with the developments of technology and the accumulation of data for the characterization of enzyme functions, artificial intelligence may become a powerful tool for the design and evolution of intelligent enzymes in the future.

Characterization of Holomorphic Functions by Zero Spherical Means

Characterization of Holomorphic Functions by Zero Spherical Means

Formulation of complementary flours from pretreated pumpkin pulp, soybeans and spinach leaves: Nutritional, functional and sensory characterization

One of the major causes of the high prevalence of young children suffering from malnutrition in developed countries is inadequate complementary feeding practices, and especially the low quality of homemade complementary foods. The present study aimed to use available local plant foods to formulate a complementary flour Which can be able to meet energy and nutrients requirements of children aged from 6 to 23 months. To achieve this goal, pumpkin was fermented, soybean soaked and roasted, and spinach steamed. The pre-treated ingredients were ground to obtain individual flours, which were blended in various proportions to obtain four complementary flours (PSS1, PSS2, PSS3, PSS4). The proximate and micronutrient composition, and the energy value of the blends were determined, and based on the results, two of them, that is; (PSS1 [Pumpkin 70 %/Soybean 25 %/Spinach 5 %], and PSS2 [Pumpkin 65 %/Soybean 25 %/Spinach 10 %]) were selected to pursue the Study. The functional properties (water absorption capacity, water solubility index, bulk density) and pasting properties of these two flours were then evaluated. Gruels were prepared from the flours and their energy densities, physical as well as sensory properties were evaluated. Moisture, ash, protein, fat, and sugar contents of PSS1 and PSS2 met the FAO/WHO standards. Fiber content in both flours was higher than the recommendation. Vitamin A and iron were sufficient in PSS2, while PSS1 had low iron content. Calcium, phosphorus, and magnesium content of PSS1 and PSS2 were significantly higher than the standards. PSS1 and PSS2 had good water absorption capacity and solubility index, with low viscosity values (213 and 173 cP respectively), interesting functional properties for complementary flours. The gruels prepared with PSS1 and PSS2 flours had good fluidity and energy densities. They were fairly appreciated based on their organoleptic characteristics, with scores of 5.96 and 5.75 for overall acceptability. PSS2 could be recommended as infant flour rich in iron, vitamin A, and protein, with good nutritional values and functional properties.

Mechanistic insights into the interaction of Lycium barbarum polysaccharide with whey protein isolate: Functional and structural characterization

Protein-polysaccharide interactions are crucial for food system structure and stability. This study investigates the interaction of Lycium barbarum polysaccharide (LBP) at 0–2.00 % concentrations with whey protein isolate (WPI), focusing on functionality and structural changes. LBP covalently grafted onto WPI, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), forming WPI-LBP complexes with a maximum degree of grafting (DG) of 44.58 % at 2.00 % LBP. This grafting reduced WPI's surface hydrophobicity (H0) and improved solubility, emulsifying properties, and digestibility under certain conditions, with optimal antioxidant activity at 1.00 % LBP. Multispectral analysis and microscopy showed LBP grafting alters WPI's secondary, tertiary, crystalline, and micro/nanostructures. The comprehensive analysis indicates that the interaction between LBP and WPI involves covalent bonding, hydrogen bonding, hydrophobic interactions, and electrostatic forces, as supported by zeta potential and chemical forces results. These findings suggest LBP-protein complexes as promising food materials for enhancing functionality and stability in the food industry.

Transcriptomic profiling of the floral fragrance biosynthesis of Iris germanica ‘Harvest of Memories’ and functional characterization of the IgTPS14 gene

Iris (Iris germanica) is a very popular ornamental plant and is known for the precious spice irone produced from its roots. Many iris varieties can also release fragrances through their flowers. However, the composition of iris aroma and the molecular mechanism of its synthesis have not been reported. In this study, we analyzed the volatile floral compounds of I. germanica ‘Harvest of Memories’ at different stages and in different tissues through headspace solid-phase microextraction gas chromatography-mass spectrometry. A total of 36 volatile compounds were identified, and linalool was the dominant component. Transcriptome analysis showed that 35 differentially expressed genes and 218 differentially expressed transcription factors were positively correlated with linalool release. According to the results of qRT-qPCR, the expression level of the IgTPS14 gene was consistent with the release trend of linalool, suggesting that IgTPS14 may play a certain role in linalool synthesis. Phylogenetic analysis showed that the IgTPS14 protein belonged to the TPS-g subfamily. An in vitro enzymatic assay of IgTPS14 and its transient and stable overexpression in tobacco indicated that this gene produced linalool. The transient silencing of IgTPS14 in petals by virus-induced gene silencing technology revealed a significant reduction in the release of linalool. These results will help explore and reveal the molecular mechanism of monoterpenoid synthesis and provide a certain reference for studying the formation of iris aroma.

Identification and functional characterisation of DNA methylation differences between East- and West-originating Finns

ABSTRACT Eastern and Western Finns show a striking difference in coronary heart disease-related mortality; genetics is a known contributor for this discrepancy. Here, we discuss the potential role of DNA methylation in mediating the discrepancy in cardiometabolic disease-risk phenotypes between the sub-populations. We used data from the Young Finns Study (n = 969) to compare the genome-wide DNA methylation levels of East- and West-originating Finns. We identified 21 differentially methylated loci (FDR < 0.05; Δβ >2.5%) and 7 regions (smoothed FDR < 0.05; CpGs ≥ 5). Methylation at all loci and regions associates with genetic variants (p < 5 × 10−8). Independently of genetics, methylation at 11 loci and 4 regions associates with transcript expression, including genes encoding zinc finger proteins. Similarly, methylation at 5 loci and 4 regions associates with cardiometabolic disease-risk phenotypes including triglycerides, glucose, cholesterol, as well as insulin treatment. This analysis was also performed in LURIC (n = 2371), a German cardiovascular patient cohort, and results replicated for the association of methylation at cg26740318 and DMR_11p15 with diabetes-related phenotypes and methylation at DMR_22q13 with triglyceride levels. Our results indicate that DNA methylation differences between East and West Finns may have a functional role in mediating the cardiometabolic disease discrepancy between the sub-populations.

Functional characterization of PIEZO1 in distinct macrophage populations.

Functional characterization of PIEZO1 in distinct macrophage populations.

Biochemical and Functional Characterization of Kombucha tea or Probiotic bacteria and Their Preservative Action on Chicken Meat

Biochemical and Functional Characterization of Kombucha tea or Probiotic bacteria and Their Preservative Action on Chicken Meat

Abstract P427: Novel SNVs of NBCe1, D405E and I803M mutants, reduced cell-surface expression and transport activity.

Introduction: The electrogenic Na + /HCO 3 - cotransporter NBCe1 is mainly expressed in proximal renal tubule, and regulate pH homeostasis and plasma volume. NBCe1 homozygous mutation causes many symptoms, including severe hypotension, proximal renal tubular acidosis (RTA), and extrarenal phenotype (migraine, mental retardation, mineral bone disorder and ocular abnormalities). Hypothesis: Undetermined single nucleotide variants (SNVs) in NBCe1 will be crucial role of sodium and bicarbonate transport. Methods: We identified the missense mutations (D405E and I803M) in NBCe1 variant A (kidney type) from the NCBI database. We conducted a comparative analysis of cellular localization and cell protein expression using confocal microscopy and western blotting. Furthermore, we performed functional analysis in D405E and I803M SNVs using two-electrode voltage clamp method. Results: Immunofluorescence analysis with confocal microscopy revealed that the D405E and I803M variants were present predominantly in the cell-surface in HEK293 cells. Western blotting in HEK293 cells confirmed that the D405E and I803M variants were significantly reduced protein expression level (25% in D405E and 57% in I803M) compared with WT, respectively. Moreover, electrophysiological analysis revealed that D405E and I803M variants were significantly reduced transport activity. These consisting data suggest that cell-expression level and transport activity are comparable. Conclusion: These data illustrate the diverse physiological consequences of distinct SNVs and underscore the importance of functional characterization in membrane transport proteins.

Functional Characterization of 11 Tentative Microneme Proteins in Type I RH Strain of Toxoplasma gondii Using the CRISPR-Cas9 System.

Toxoplasma gondii, a pathogenic apicomplexan parasite, infects approximately one third of the world's population and poses a serious threat to global public health. Microneme proteins (MICs) secreted by the microneme, an apical secretory organelle of T. gondii, play important roles in the invasion, motility, and intracellular survival of T. gondii. In this study, we selected 11 genes of interest (GOIs) of T. gondii, tentative MICs predicted to be localized in micronemes, and we used the CRISPR-Cas9 system to construct epitope tagging strains and gene knockout strains to explore the localization and function of these 11 tentative MICs. Immunofluorescence assay showed that nine tentative MICs (TGME49_243930, TGME49_200270, TGME49_273320, TGME49_287040, TGME49_261710, TGME49_205680, TGME49_304490, TGME49_245485, and TGME49_224620) were localized or partially localized in the microneme, consistent with the prediction. However, TGME49_272380 and TGME49_243790 showed different localizations from the prediction, being localized in the endoplasmic reticulum and the dense granule, respectively. Further functional characterization of the 11 RHΔGOI strains revealed that deletion of these 11 GOIs had no significant effect on plaque formation, intracellular replication, egress, invasion ability, and virulence of T. gondii. Although these 11 GOIs are not essential genes for the growth and virulence of tachyzoites of type I RH strain, they may have potential roles in other developmental stages or other genotypes of T. gondii. Thus, further research should be performed to explore the possible role of the nine mics and the other two GOIs in other life cycle stages and other genotypes of T. gondii.

Fully defined NGN2 neuron protocol reveals diverse signatures of neuronal maturation

NGN2-driven induced pluripotent stem cell (iPSC)-to-neuron conversion is a popular method for human neurological disease modeling. In this study, we present a standardized approach for generating neurons utilizing clonal, targeted-engineered iPSC lines with defined reagents. We demonstrate consistent production of excitatory neurons at scale and long-term maintenance for at least 150days. Temporal omics, electrophysiological, and morphological profiling indicate continued maturation to postnatal-like neurons. Quantitative characterizations through transcriptomic, imaging, and functional assays reveal coordinated actions of multiple pathways that drive neuronal maturation. We also show the expression of disease-related genes in these neurons to demonstrate the relevance of our protocol for modeling neurological disorders. Finally, we demonstrate efficient generation of NGN2-integrated iPSC lines. These workflows, profiling data, and functional characterizations enable the development of reproducible human invitro models of neurological disorders.

Functional characterization of a novel lipoxygenase from Aliikangiella coralliicola for natural modification on gelation of soy protein isolate

Lipoxygenase (LOX) can catalyze the production of hydroperoxides from polyunsaturated fatty acids, offering promising applications in the food industries with especial contribution of naturally modified function for food gelation. To date, commercially available LOXs primarily sourced from soybean meal, facing challenges such as containing isoenzymes and exhibiting low catalytic activity for broad utilization restrictions. Herein, a LOX was first derived from the marine microorganism Aliikangiella coralliicola (AcLOX) with a molecular weight of 80 kDa expressed in Escherichia coli. AcLOX exhibited good stability over a wide pH range of 4.0–9.0. Additionally, AcLOX displayed specific activity against α-Linolenic acid (ALA) of 6.67 × 104 U/mg, and kinetic parameters values for Km, kcat, and kcat/Km of 0.58 μM, 1.40 s−1, and 2.42 μM−1 s−1, respectively. Moreover, Ca2+ and dimethyl sulfoxide (DMSO) activated the activity of AcLOX. To explore its gelation modification ability, AcLOX was applied to induce the cross-linking of soy protein isolate (SPI) subunits with increased particle size, potential, surface hydrophobicity (So), and as well as gel hardness at appropriate concentrations. This is the inaugural study to unveil the gelation modification ability of LOX from Aliikangiella coralliicola. These findings provide superior enzymatic performance and cross-linking potential of AcLOX for industrial applications.

Comprehensive genome-wide analysis of PUB (Plant U-box) genes in rye and its closely related species and functional characterization of ScPUB6

Comprehensive genome-wide analysis of PUB (Plant U-box) genes in rye and its closely related species and functional characterization of ScPUB6

Identification of Shaker Potassium Channel Family Members and Functional Characterization of SsKAT1.1 in Stenotaphrum secundatum Suggest That SsKAT1.1 Contributes to Cold Resistance.

Stenotaphrum secundatum is an excellent shade-tolerant warm-season turfgrass. Its poor cold resistance severely limits its promotion and application in temperate regions. Mining cold resistance genes is highly important for the cultivation of cold-resistant Stenotaphrum secundatum. Although there have been many reports on the role of the Shaker potassium channel family under abiotic stress, such as drought and salt stress, there is still a lack of research on their role in cold resistance. In this study, the transcriptome database of Stenotaphrum secundatum was aligned with the whole genome of Setaria italica, and eight members of the Shaker potassium channel family in Stenotaphrum secundatum were identified and named SsKAT1.1, SsKAT1.2, SsKAT2.1, SsKAT2.2, SsAKT1.1, SsAKT2.1, SsAKT2.2, and SsKOR1. The KAT3-like gene, KOR2 homologous gene, and part of the AKT-type weakly inwardly rectifying channel have not been identified in the Stenotaphrum secundatum transcriptome database. A bioinformatics analysis revealed that the potassium channels of Stenotaphrum secundatum are highly conserved in terms of protein structure but have more homologous members in the same group than those of other species. Among the three species of Oryza sativa, Arabidopsis thaliana, and Setaria italica, the potassium channel of Stenotaphrum secundatum is more closely related to the potassium channel of Setaria italica, which is consistent with the taxonomic results of these species belonging to Paniceae. Subcellular location experiments demonstrate that SsKAT1.1 is a plasma membrane protein. The expression of SsKAT1.1 reversed the growth defect of the potassium absorption-deficient yeast strain R5421 under a low potassium supply, indicating that SsKAT1.1 is a functional potassium channel. The transformation of SsKAT1.1 into the cold-sensitive yeast strain INVSC1 increased the cold resistance of the yeast, indicating that SsKAT1.1 confers cold resistance. The transformation of SsKAT1.1 into the salt-sensitive yeast strain G19 increased the resistance of yeast to salt, indicating that SsKAT1.1 is involved in salt tolerance. These results suggest that the manipulation of SsKAT1.1 will improve the cold and salt stress resistance of Stenotaphrum secundatum.

Genome-wide investigation of the nuclear factor Y gene family in Ginger (Zingiber officinale Roscoe): evolution and expression profiling during development and abiotic stresses

BackgroundNuclear factor Y (NF-Y) plays a vital role in numerous biological processes as well as responses to biotic and abiotic stresses. However, its function in ginger (Zingiber officinale Roscoe), a significant medicinal and dietary vegetable, remains largely unexplored. Although the NF-Y family has been thoroughly identified in many plant species, and the function of individual NF-Y TFs has been characterized, there is a paucity of knowledge concerning this family in ginger.MethodsWe identified the largest number of NF-Y genes in the ginger genome using two BLASTP methods as part of our ginger genome research project. The conserved motifs of NF-Y proteins were analyzed through this process. To examine gene duplication events, we employed the Multiple Collinearity Scan toolkit (MCScanX). Syntenic relationships of NF-Y genes were mapped using the Dual Synteny Plotter software. Multiple sequence alignments were performed with MUSCLE under default parameters, and the resulting alignments were used to generate a maximum likelihood (ML) phylogenetic tree with the MEGA X program. RNA-seq analysis was conducted on collected samples, and statistical analyses were performed using Sigma Plot v14.0 (SYSTAT Software, USA).ResultsIn this study, the ginger genome was utilized to identify 36 NF-Y genes (10 ZoNF-YAs, 16 ZoNF-YBs, and 10 ZoNF-YCs), which were renamed based on their chromosomal distribution. Ten distinct motifs were identified within the ZoNF-Y genes, with certain unique motifs being vital for gene function. By analyzing their chromosomal location, gene structure, conserved protein motifs, and gene duplication events, we gained a deeper understanding of the evolutionary characteristics of these ZoNF-Y genes. Detailed analysis of ZoNF-Y gene expression patterns across various tissues, performed through RNA-seq and qRT-PCR, revealed their significant role in regulating ginger rhizome and flower growth and development. Additionally, we identified the ZoNF-Y family genes that responded to abiotic stresses.ConclusionThis study represents the first identification of the ZoNF-Y family in ginger. Our findings contribute to research on evolutionary characteristics and provide a better understanding of the molecular basis for development and abiotic stress response. Furthermore, it lays the foundation for further functional characterization of ZoNF-Y genes with an aim of ginger crop improvement.

Structural and functional characterization of gamma-irradiation-enhanced nanocomposites for colorectal cancer therapy

The purpose of this research was to develop and evaluate a nanocomposite material for the treatment of colorectal cancer. The nanocomposite consisted of manganese-doped silver nanoparticles coated with silica (Si-Ag: Mn3O4). The nanocomposite was synthesized by co-precipitating silver and manganese oxide, followed by a silica coating using the Stöber method. Extensive characterization was conducted to assess the morphological, optical, and stability properties. The key findings of this study are the Si-Ag: Mn3O4 nanocomposite exhibited unexpected and potent cytotoxicity against colorectal cancer cells, exceeding the effects of the positive control, cisplatin. The silica coating increased the Ag: Mn ratio, further enhancing the efficacy of the nanocomposite in cancer treatment; the nanocomposite maintained cellular mobility without agglomeration or sedimentation, indicating its potential for targeted tumor destruction; and gamma irradiation of the nanocomposite further increased its cytotoxic effects on colorectal cancer cells. In conclusion, the unique combination of silver, manganese oxide, and silica in this nanocomposite framework shows great promise for advanced applications in selective cancer therapy. The nanocomposite demonstrated significant cytotoxicity against HT-29 colorectal cancer cells, particularly at a gamma dose of 5.256 kGy. The results of this study open new perspectives in biological research, cancer treatment, and nanomedicine.

Dissecting the Enterococcal Polysaccharide Antigen (EPA) structure to explore innate immune evasion and phage specificity

Streptococci, Lactococci and Enterococci all produce L-rhamnose-containing cell wall polysaccharides which define Lancefield serotypes and represent promising candidates for the design of glycoconjugate vaccines. The L-rhamnose containing Enterococcal Polysaccharide Antigen (EPA), produced by the opportunistic pathogen Enterococcus faecalis, plays a critical role in normal growth, division, biofilm formation, antimicrobial resistance, phage susceptibility, and innate immune evasion. Despite the critical role of this polymer in E. faecalis physiology and host-pathogen interactions, little information is available on its structure and biosynthesis. Here, using an NMR approach, we elucidate the structure of EPA and propose a model for biosynthesis. We report the structure of the EPA-peptidoglycan linkage unit and reveal an unprecedented complexity of the EPA rhamnose backbone and decoration subunits. Finally, we explore the impact of several EPA structural modifications on innate immune evasion and recognition by bacteriophages. This work represents a first step towards the functional characterisation of EPA and the rational design of therapeutic strategies against a group of important pathogens.

Genome-Wide Identification of NAC Family Genes and Their Expression Analyses in Response to Osmotic Stress in Cannabis sativa L.

NAC (NAM, ATAF1/2, and CUC2) transcription factors are unique and essential for plant growth and development. Although the NAC gene family has been identified in a wide variety of plants, its chromosomal location and function in Cannabis sativa are still unknown. In this study, a total of 69 putative CsNACs were obtained, and chromosomal location analysis indicated that the CsNAC genes mapped unevenly to 10 chromosomes. Phylogenetic analyses showed that the 69 CsNACs could be divided into six subfamilies. Additionally, the CsNAC genes in group IV-a are specific to Cannabis sativa and contain a relatively large number of exons. Promoter analysis revealed that most CsNAC promoters contained cis-elements related to plant hormones, the light response, and abiotic stress. Furthermore, transcriptome expression profiling revealed that 24 CsNAC genes in two Cannabis sativa cultivars (YM1 and YM7) were significantly differentially expressed under osmotic stress, and these 12 genes presented differential expression patterns across different cultivars according to quantitative real-time PCR (RT-qPCR) analysis. Among these, the genes homologous to the CsNAC18, CsNAC24, and CsNAC61 genes have been proven to be involved in the response to abiotic stress and might be candidate genes for further exploration to determine their functions. The present study provides a comprehensive insight into the sequence characteristics, structural properties, evolutionary relationships, and expression patterns of NAC family genes under osmotic stress in Cannabis sativa and provides a basis for further functional characterization of CsNAC genes under osmotic stress to improve agricultural traits in Cannabis sativa.

Biallelic variants in α-tubulin isotypes cause female infertility characterised as recurrent preimplantation embryo arrest

BackgroundRecurrent preimplantation embryo developmental arrest (RPEA) is the most common phenotype in assisted reproductive technology treatment failure associated with identified genetic abnormalities. Currently known maternal genetic variants explain only a...