Bound Form Research Articles

Use of multivariant enzyme-linked immunosorbent assay (ELISA) in the diagnosis of autoimmune bullous disorders in a resource-limited setting: A single-center experience

Background Autoimmune blistering disorders (AIBD) result from the formation of auto-antibodies against adhesion proteins of the skin/mucosa(e). These auto-antibodies can be detected in the bound form in the tissue using direct immunofluorescence (DIF) or blood circulation using enzyme-linked immunosorbent assay (ELISA) or other methods. Objectives The objective of this study was to evaluate the concordance rate between the results of multivariant ELISA and the diagnosis of AIBD made using DIF and histopathology in an appropriate clinical context. Methods This was a retrospective study (December 2020 to April 2023) in which the multivariant ELISA assay (able to detect antibodies against desmoglein 1, desmoglein 3, BP180, BP230, envoplakin, and collagen VII) data were retrieved from the dermatology laboratory. Corresponding clinical and histopathology data were searched from relevant institutional databases. As per routine practice, the final diagnosis was assigned based on the clinical presentation, histopathology features and corresponding DIF report. Results After screening the records of 338 patients during the study period, 253 patients were included. Of them, 194 had AIBD and 59 had non-AIBD. In the autoimmune blistering disorder group, 122 and 72 patients had pemphigus and pemphigoid, respectively. Overall, a good level of agreement was found between multivariant ELISA results and the final diagnosis (Fleiss kappa = 0.631, p-value < 0.001). The pemphigus vulgaris group exhibited good agreement (kappa = 0.796, p < 0.001), while pemphigus foliaceous, bullous pemphigoid and non-autoimmune blistering disorders demonstrated moderate agreement (kappa = 0.641, 0.651, 0.533, respectively; p < 0.001). The mucous membrane pemphigoid group had a fair agreement (kappa = 0.289; p < 0.001). Limitations The limitations for the study were its retrospective design, fewer number of patients in certain groups like paraneoplastic pemphigus and gold-standard single antigen specific ELISA was not done. Conclusion Considering good agreement between the multivariant ELISA and the gold-standard diagnosis (clinical findings plus histopathology plus DIF), multivariant ELISA can be used for the diagnosis of AIBDs in places where facilities for DIF are unavailable. Multivariant ELISA can improve etiological diagnosis for a set of autoimmune blistering disorders whose target antigens are represented in the multivariant panel.

Phenolic Acids and Their Relationship to Nutritional and Technological Grain Parameters of Durum Wheat Under Variable Treatment Intensity in Central European Conditions

The objective of this two-year study was primarily the evaluation of the free and the bound forms of phenolic acids and phenolic aldehydes (PAAs) in grains of four selected cultivars of spring durum wheat subjected to three treatment intensities (GD—Green Deal, BT—Basic and IT—Intensive). All treatments included a common basic level and different spring production levels of nitrogen fertilisation (0 kg N in the case of GD; 30 kg N in the case of BT; and 60 kg N in the case of IT). Pesticide applications included herbicides and insecticides in both the BT and IT treatments, which were supplemented by combinations of fungicide and morphoregulator in the IT treatment. The GD treatment included only basic nitrogen, herbicide protection, and the application of a biostimulator (ExelGrow). The spring durum wheat cultivars subjected to testing were cultivated under Central European conditions, specifically in the Czech Republic’s central Bohemian region. UHPLC-ESI-MS/MS was used for the detection and accurate quantification of PAAs. In parallel, 12 other nutritional and basic technological parameters of the cereal were evaluated. Nine bound and seven free forms of PAAs were quantified in the analysed cereal samples. Bound forms of PAAs were dominant, accounting for 99.4% of total PAAs. Considering single PAAs, ferulic acid was the most abundant, accounting for 87% of the total bound PAAs. Interestingly, year and treatment intensity were the key factors in the variability of both free and bound PAAs, but these factors had different effects on bound PAAs. Under low nitrogen conditions, plants responded with an increase in free PAAs in particular, as well as in three bound PAAs. Unfavourable weather conditions, combined with the presence of biotic factors (e.g., Fusarium infections), significantly influenced the increase in both PAA groups, with the exception of free p-coumaric acid. PCA analysis confirmed close relationships between PAAs within both categories (free and bound). Subsequent correlation analysis further revealed that the immunoreactive gluten component (G12) exhibited a high negative correlation with the dominant ferulic acid (r = −0.70) and sinapic acid (r = −0.68). Additionally, moderate negative correlations were observed between four bound phenolic acids and grain hardness (r = −0.48–−0.60).

Unlocking the role of novel primary/di-amine oxidases in crop improvement: Tissue specificity leads to specific roles connected to abiotic stress, hormone responses and sensing nitrogen

Genetic improvements of solanaceous crops for quality and stress responsive traits are needed because of the central role vegetables and fruits have in providing nutrients to human diets. Copper amine oxidase (CuAO) encoding genes involved in metabolism of primary/di-amine nitrogenous compounds, play a role in balancing internal nitrogen (N) pools especially when external N supply fluctuates during growth, development and environmental stresses. In the present study, we investigated the occurrence, molecular evolution and possible role(s) of these unknown genes in tomato crops. Multiple genome-wide bioinformatics approaches led to the identification of eight bona fide CuAO genes (SlCuAO1–SlCuAO8) in the tomato genome with gene numbers like those in Arabidopsis and rice indicating their conserved functional relevance with a tandemly duplicated SlCuAO6-SlCuAO7 pair at chr.9. A conserved intron-exon size and phase distribution for SlCuAO2, 3, 4 pairs are similar to a recently identified single duckweed SpCuAO1 orthologue gene indicating its evolutionary conservation. Synteny analysis showed their closest association to Arabidopsis and but not with rice. Transcriptome data indicated that gene expression for about six genes (SlCuAO1, 2, 3, 4, 6, 7) is root specific, fruit specific for SlCuAO5 and flower specific for SlCuAO8 thus indicating amine oxidation is variable across tissues with a prominance in the root tissue. The majority of CuAO genes are negatively regulated by methyl jasmonate. Positive regulation, however, involves CuAO3/8. Transcript analysis of the ethylene-deficient transgenic lines indicated that ethylene is required for activation of SlCuAO4. CuAO4 and CuAO5 exhibited most significant tissues-independent gene expression responses across various nitrogen regimes. Drought, heat and N stress identified CuAO5 as an overlapping highly expressed gene that corroborates with putrescine accumulation for free and conjugated forms with an opposite abundance of bound forms. Taken together our study highlights new insights into the roles of copper amine oxidation genes and identifies CuAO5 as a multiple stress induced gene that can be used in genetic improvement programs for combining heat, drought and nitrogen use efficiency related traits.

The loss-of-function of AtNATA2 enhances AtADC2-dependent putrescine biosynthesis and priming, improving growth and salinity tolerance in Arabidopsis.

Putrescine (Put) is a promising small molecule-based biostimulant to enhance plant growth and resilience, though its mode of action remains unclear. This study investigated the Put priming effect on Arabidopsis mutant lines (Atadc1, Atadc2, Atnata1, and Atnata2) under control conditions and salinity to understand its role in regulating plant growth. The Atadc2 mutant, characterized by reduced endogenous Put levels, showed insensitivity to Put priming without growth enhancement, which was linked to significant imbalances in nitrogen metabolism, including a high Gln/Glu ratio. Contrarily, the Atnata2 mutant exhibited significant growth improvement and upregulated AtADC2 expression, particularly under Put priming, highlighting these genes' involvement in regulating plant development. Put priming enhanced plant growth by inducing the accumulation of specific polyamines (free, acetylated, conjugated, or bound form) and improving light-harvesting efficiency, particularly in the Atnata2 line. Our findings suggest that AtNATA2 may negatively regulate Put synthesis and accumulation via AtADC2 in the chloroplast, impacting light harvesting in photosystem II (PSII). Furthermore, the Atadc2 mutant line exhibited upregulated AtADC1 but reduced AcPut levels, pointing to a cross-regulation among these genes. The regulation by AtNATA2 on AtADC2 and AtADC2 on AtADC1 could be crucial for plant growth and overall stress tolerance by interacting with polyamine catabolism, which shapes the plant metabolic profile under different growth conditions. Understanding the regulatory mechanisms involving crosstalk between AtADC and AtNATA genes in polyamine metabolism and the connection with certain SMBBs like Put can lead to more effective agricultural practices, improving plant growth, nitrogen uptake, and resilience under challenging conditions.

Free and bound phenolic profiles and antioxidant ability of eleven marine macroalgae from the South China Sea.

Marine macroalgae are of broad interest because of their abundant bioactive phenolic compounds. However, only a few previous studies have focused on bound phenolic compounds. In this study, there were significant differences in total phenolic content, total phlorotannin content, total flavonoid content, and antioxidant ability in free and bound forms, as well as in their bound-to-free ratios, among 11 marine macroalgal species from the South China Sea. Padina gymnospora had the highest total phenolic content of free fractions, and total phlorotannin content, total flavonoid content, and antioxidant activity of free fractions. Sargassum thunbergii had the highest total phlorotannin content of bound fractions, whereas Sargassum oligocystum had the highest total flavonoid content and total phenolic content of bound fractions. Moreover, 15 phenolic acids, 35 flavonoids, 2 stilbenes, 3 bromophenols, and 3 phlorotannins were characterized and quantified using ultra-high-performance liquid chromatography with Xevo triple quadrupole mass spectrometry, and 42 phenolic compounds were reported in the bound fractions of seaweeds for the first time. Among the species, the number and amount of free and bound phenolic compounds varied greatly and the main components were different. Padina gymnospora had the largest total phenolic number, while Turbinaria ornata showed the highest total phenolic amount. Coutaric acid and diosmetin were dominant in Sargassum polycystum, and hinokiflavone was dominant in Caulerpa lentillifera, and cyanidin was dominant in the other seaweeds. Hierarchical cluster analysis was used to divide the seaweed species into seven groups. This study revealed that Padina gymnospora, Sargassum thunbergii, Turbinaria ornata, and Sargassum oligocystum are promising functional food resources.

The effects of milling on the chemical forms, bioavailability, and in vivo distribution of Vitamin B1 in rice

Milling not only reduces the content of Vitamin B1 (Vit B1) in rice but may even affect its bioaccessibility, further affecting its activity in vivo. Therefore, this study investigated the changes in Vit B1 content and its chemical forms in rice at different degrees of milling (DOM) before and after cooking, as well as their bioaccessibility in vitro and bioavailability in vivo. In raw unmilled rice, the proportion of bound Vit B1 was slightly lower than that of free Vit B1, and the proportion increased significantly after cooking. DOM< 8% hindered this change of bound Vit B1 after cooking, resulting in a decrease in Vit B1 thermal stability from 90% to 80% and bioaccessibility from 80% to 60%. Fortunately, the content of Vit B1 stabilized in the digestive fluid remained superior to that of the high DOM group. DOM<8% shortened the time for Vit B1 to peak in the blood concentration of mice, whereas the decrease in bioavailability of Vit B1 in blood and liver was insignificant, unlike bioaccessibility. Hence, DOM < 8% can be applied to the rice milling process to maintain nutritional value. Correlation analyses indicated that the bound form of Vit B1 in cooked rice was the main factor affecting its digestion and absorption. These findings provide further support for the development of moderate rice processing.

PerR: A Peroxide Sensor Eliciting Metal Ion-dependent Regulation in Various Bacteria.

Bacteria have to thrive in difficult conditions wherein their competitors generate partially reduced forms of oxygen, like hydrogen peroxide and superoxides. These oxidative stress molecules can also arise from within via the autoxidation of redox enzymes. To adapt to such conditions, bacteria express detox enzymes as well as repair proteins. Transcription factors regulate these defenses, and PerR is one of them. PerR is a Fur family transcriptional regulator that senses peroxide stress. Metal-bound PerR (either Mn2+ or Fe2+) can repress transcription of its regulon, but only the Fe2+-bound form of PerR can sense H2O2. This review describes different aspects of PerR and its varied roles, specifically in bacterial pathogens. Despite having roles beyond sensing peroxides, it is an underrated regulator that needs to be explored more deeply in pathogens.

Structural basis for CCR6 modulation by allosteric antagonists

The CC chemokine receptor 6 (CCR6) is a potential target for chronic inflammatory diseases. Previously, we reported an active CCR6 structure in complex with its cognate chemokine CCL20, revealing the molecular basis of CCR6 activation. Here, we present two inactive CCR6 structures in ternary complexes with different allosteric antagonists, CCR6/SQA1/OXM1 and CCR6/SQA1/OXM2. The oxomorpholine analogues, OXM1 and OXM2 are highly selective CCR6 antagonists which bind to an extracellular pocket and disrupt the receptor activation network. An energetically favoured U-shaped conformation in solution that resembles the bound form is observed for the active analogues. SQA1 is a squaramide derivative with close-in analogues reported as antagonists of chemokine receptors including CCR6. SQA1 binds to an intracellular pocket which overlaps with the G protein site, stabilizing a closed pocket that is a hallmark of inactive GPCRs. Minimal communication between the two allosteric pockets is observed, in contrast to the prevalent allosteric cooperativity model of GPCRs. This work highlights the versatility of GPCR antagonism by small molecules, complementing previous knowledge of CCR6 activation, and sheds light on drug discovery targeting CCR6.

Supramolecular complexes of GCAP1: implications for inherited retinal dystrophies

Guanylate Cyclase Activating Protein 1 (GCAP1) is a calcium sensor that regulates the enzymatic activity of retinal Guanylate Cyclase 1 (GC1) in photoreceptors in a Ca2+/Mg2+ dependent manner. While point mutations in GCAP1 have been associated with inherited retinal dystrophies (IRDs), their impact on protein dimerization or on the possible interaction with the potent GC1 inhibitor RD3 (retinal degeneration protein 3) has never been investigated. Here, we integrate exhaustive in silico investigations with biochemical assays to evaluate the effects of the p.(E111V) substitution, associated with a severe form of IRD, on GCAP1 homo- and hetero-dimerization, and demonstrate that wild type (WT) GCAP1 directly interacts with RD3. Although inducing constitutive activation in GC1, the E111V substitution only slightly affects the dimerization of GCAP1. Both WT- and E111V-GCAP1 are predominantly monomeric in the absence of the GC1 target, however E111V-GCAP1 shows a stronger tendency to be monomeric in the Ca2+-bound form, corresponding to GC1 inhibiting state. Reconstitution experiments performed in the co-presence of WT-GCAP1, E111V-GCAP1 and RD3 restored nearly physiological regulation of the GC1 enzymatic activity in terms of cGMP synthesis and Ca2+-sensitivity, suggesting new scenarios for biologics-mediated treatment of GCAP1-associated IRDs.

Scavenger Receptor C1 Mediates Toxicity of Binary Toxin from Lysinibacillus sphaericus to Ag55 Cells.

Lysinibacillus sphaericus harboring Binary (BinA and BinB) toxins is highly toxic against Anopheles and Culex mosquito larvae. The Anopheles Ag55 cell line is a suitable model for investigating the mode of Bin toxin action. Based on the low-levels of α-glycosidase Agm3 mRNA in Ag55 cells and the absence of detectable Agm3 proteins, we hypothesized that a scavenger receptor could be mediating Bin cytotoxicity. Preliminary RNA interference knockdown of the expressed scavenger receptors, combined with Bin cytotoxicity assays, was conducted. The scavenger Receptor C1 (SCRC1) became the focus of this study, as a putative receptor for Bin toxins in Ag55 cells, and SCRBQ2 was selected as a negative control. Open reading frames encoding SCRC1 and SCRBQ2 were cloned and expressed in vitro, and polyclonal antibodies were prepared for immunological analyses. The RNAi silencing of SCRC1 and SCRBQ2 resulted in the successful knockdown of both SCRC1 and SCRBQ2 transcripts and protein levels. The cytolytic toxicity of Bin against Ag55 cells was severely reduced after the SCRC1-RNAi treatment. The phagocytic receptor protein SCRC1 mediates endocytosis of the Bin toxin into Ag55 cells, thereby facilitating its internal cytological activity. The results support a mechanism of the Bin toxin entering Ag55 cells, possibly via SCRC1-mediated endocytosis, and encourage investigations into how Bin is transferred from its bound form on the midgut epithelial cells into the epithelial endocytic system.

The role of soil elemental forms in the soil-plant migration system: An example of heavy metals in Epimedium production areas

Investigating the heavy metal content and migration behavior in the soil and plant system of Epimedium origin is crucial for ensuring the safety and sustainable development of this valuable medicinal plant. The concentration of heavy metals in the soil of Epimedium was found to be highest in the residuel form (RS) and lowest in the water-soluble form (WS). Among the different parts of the Epimedium plant, leaves showed the highest content of Cr, Ni, Cu, As, and Pb with average contents of 0.02 mg.kg−1, 0.01 mg.kg−1, 0.11 mg.kg−1, 0.16 mg.kg−1, and 0.01 mg.kg−1, respectively, and roots showed the highest content of Zn with average contents of 0.30 mg.kg−1. The results of correlation analysis and linear fitting showed significant correlation and better linear fitting results between different parts of Epimedium (roots, stems and leaves) and various forms of heavy metals in soil. Among them, Zn (ion-exchange form) and Zn (leaf) showed the best fit with R2 reaching 0.74. Results from random forest modeling indicated that the forms of heavy metals in soil, such as bound form, ion exchange form, and strong organic bound form, significantly influenced the distribution of heavy metals in roots, stems, and leaves of Epimedium. Overall, besides the readily available heavy metals in soil, other forms of heavy metals also play a critical role in the soil-plant migration system.

Assessing the influence of extrusion processing on functional properties and phytochemical profiles in diverse rice bran varieties

AbstractBackground and ObjectivesThe aims of this study were to assess the functional and phytochemical characteristics, as well as the antioxidant properties, of bran from five distinct rice varieties. Additionally, it evaluated the impact of extrusion cooking on the composition and quantity of individual phytochemicals in both free and bound states in the bran of these varieties.FindingsThe functional properties and phytochemical profiles of raw and extruded rice bran from different rice varieties differed significantly. The extrusion process significantly influenced the color, protein solubility, foaming properties, polyphenolics, and amino acid composition of rice bran. Total phenolics ranged from 3.18 to 4.80 mg GAE/g, increased by 12.6%–17% postextrusion. Similarly, total flavonoid content ranged from 15.22 to 17.37 mg RTE/100 g, with a 30%–33% increase after extrusion cooking. Throughout the extrusion process, a rise in the concentration of free phenolics accompanied by a decrease in the concentration of bound phenolics was observed.ConclusionThe study revealed the impact of extrusion cooking on the distribution of total phytochemicals, particularly phenolics and flavonoids, and the levels of antioxidant activity in free and bound phenolics, as well as amino acid composition and functional properties. The extrusion cooking increased levels of free phenolics and 2,2‐diphenyl‐1‐picrylhydrazyl activity, while bound forms decreased. Extrusion cooking variably impacted the functionality and phytochemical content of bran from different rice varieties, thus influencing their likely applications.Significance and NoveltyThis study established the foundation for producing food products using extruded rice bran, offering health benefits and meeting the growing demand in the functional food sector.

Impact of traditional and innovative cooking techniques on Italian black rice (Oryza sativa L., Artemide cv) composition

Due to its high polyphenol content, black rice plays a significant role in good nutrition; however, these antioxidant compounds are affected by heat treatments required for the rice consumption. The aim of this work was to investigate how cooking affects the composition of Artemide black rice, comparing innovative methods, such as sous vide, with traditional domestic techniques (risotto and pilaf). Proteins and ashes were not affected by cooking, except for pilaf rice, where a 42 % ashes decrease was observed; fiber content increased after all cooking methods, reaching a 29 % increase in the risotto. Antioxidant activity, total polyphenols, anthocyanins and proanthocyanidins were reduced on average of 40 %, 34 %, 43 % and 39 %, respectively. Individual anthocyanins decreased, while phenolic acids and other flavonoids presented different behaviours, also depending if considered in their free or bound form. Cyanidin-3-O-glucoside was reduced up to 56 % in the sous vide cooked rice at 99 °C, and only by 45 % and 37 % in the risotto and sous vide cooked rice at 89 °C, respectively. Traditional risotto preparation and the innovative sous vide cooking at 89 °C also maintained the highest antioxidant polyphenols content, saving 63 % of the antioxidant activity in respect to the raw black rice. Concluding, these last techniques can be suggested for a better preservation of bioactive compounds.

Deciphering GB1's Single Mutational Landscape: Insights from MuMi Analysis.

Mutational changes that affect the binding of the C2 fragment of Streptococcal protein G (GB1) to the Fc domain of human IgG (IgG-Fc) have been extensively studied using deep mutational scanning (DMS), and the binding affinity of all single mutations has been measured experimentally in the literature. To investigate the underlying molecular basis, we perform in silico mutational scanning for all possible single mutations, along with 2 μs-long molecular dynamics (WT-MD) of the wild-type (WT) GB1 in both unbound and IgG-Fc bound forms. We compute the hydrogen bonds between GB1 and IgG-Fc in WT-MD to identify the dominant hydrogen bonds for binding, which we then assess in conformations produced by Mutation and Minimization (MuMi) to explain the fitness landscape of GB1 and IgG-Fc binding. Furthermore, we analyze MuMi and WT-MD to investigate the dynamics of binding, focusing on the relative solvent accessibility of residues and the probability of residues being located at the binding interface. With these analyses, we explain the interactions between GB1 and IgG-Fc and display the structural features of binding. In sum, our findings highlight the potential of MuMi as a reliable and computationally efficient tool for predicting protein fitness landscapes, offering significant advantages over traditional methods. The methodologies and results presented in this study pave the way for improved predictive accuracy in protein stability and interaction studies, which are crucial for advancements in drug design and synthetic biology.

Simulation of Soluble and Bound VEGF-stimulated in vitro Capillary-like Network Formation on Deformed Substrate.

Capillary plexus cultivation is crucial in tissue engineering and regenerative medicine. Theoretical simulations have been conducted to supplement the expensive experimental works. However, the mechanisms connecting mechanical and chemical stimuli remained undefined, and the functions of the different VEGF forms in the culture environment were still unclear. In this paper, we developed a hybrid model for simulating short-term in vitro capillary incubations. We used the Cellular Potts model to predict individual cell migration, morphology change, and continuum mechanics to quantify biogel deformation and VEGF transport dynamics. By bridging the mechanical regulation and chemical stimulation in the model, the results showed good agreement between the predicted network topology and experiments, in which elongated cells connected, forming the network cords and round cells gathered, creating cobblestone-like aggregates. The results revealed that the capillary-like networks could develop in high integrity only when the mechanical and chemical couplings worked adequately, with the cell morphology and haptotaxis driven by the soluble and bound forms of VEGF, respectively, functioning simultaneously.

Reversion induced LIM domain protein (RIL) is a Daam1-interacting protein and regulator of the actin cytoskeleton during non-canonical Wnt signaling

The Daam1 protein regulates Wnt-induced cytoskeletal changes during vertebrate gastrulation though its full mode of action and binding partners remain unresolved. Here we identify Reversion Induced LIM domain protein (RIL) as a new interacting protein of Daam1. Interaction studies uncover binding of RIL to the C-terminal actin-nucleating portion of Daam1 in a Wnt-responsive manner. Immunofluorescence studies showed subcellular localization of RIL to actin fibers and co-localization with Daam1 at the plasma membrane. RIL gain- and loss-of-function approaches in Xenopus produced severe gastrulation defects in injected embryos. Additionally, a simultaneous loss of Daam1 and RIL synergized to produce severe gastrulation defects indicating RIL and Daam1 may function in the same signaling pathway. RIL further synergizes with another novel Daam1-interacting protein, Formin Binding Protein 1 (FNBP1), to regulate gastrulation. Our studies altogether show RIL mediates Daam1-regulated non-canonical Wnt signaling that is required for vertebrate gastrulation.

Screening of the Medicinal Plant-Based Metabolites Responsible for Silver Nanoparticle Phytofabrication

In this study, the detailed phytochemical composition of two medicinal plants was investigated and their importance in silver nanoparticles (AgNPs) synthesis was highlighted. Aqueous extracts from the leaves of both plants were used for various analytical techniques, including UV-vis spectroscopy, Gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared spectroscopy (FTIR) and Thin layer chromatography (TLC). Quantification of total phenolics and flavonoids in different fractions of plant extracts revealed significant concentrations. TLC profiling showed a higher abundance of free phenolic and flavonoid compounds compared to bound forms in both plants, suggesting that they contribute significantly to total phenolic and flavonoid content. UV-vis spectra from 200 to 600 nm revealed the presence of aromatic rings and chromophores in leaf extracts. Furthermore, the GC-MS analysis identified several bioactive compounds, some of which were found to be common between these two species. The results demonstrate that various bioactive compounds such as phenols, flavonoids, alkaloids, carotenoids and terpenoids were present in these plant species. These compounds efficiently serve as both reducing and stabilizing agents during phytofabrication of AgNPs, consequently eliminating the need for the use of hazardous chemicals.

Formin Binding Protein 1 (FNBP1) regulates non-canonical Wnt signaling and vertebrate gastrulation

The Formin protein Daam1 is required for Wnt-induced cytoskeletal changes during gastrulation, though how it accomplishes this remains unresolved. Here we report the characterization of Formin Binding Protein 1 (FNBP1) as a binding partner of Daam1. The interaction of Daam1 with FNBP1 and its domains required for this interaction were delineated. Immunofluorescence studies showed FNBP1 co-localizes with Daam1, and is an integral component of the actin cytoskeletal complex that is responsive to Wnt stimulation. Specifically, FNBP1 can induce intracellular tubule-like structures and localize to focal adhesions suggesting a role for FNBP1 in cell migration. Functional FNBP1 studies in Xenopus embryos uncover a critical role for FNBP1 in regulating vertebrate gastrulation. Additionally, suboptimal doses of Daam1 and FNBP1 synergize to produce severe gastrulation defects, indicating FNBP1 and Daam1 may function within the same signaling pathway. These results together show FNBP1 is an integral component of Daam1-regulated non-canonical Wnt signaling required for vertebrate gastrulation.

Liberated bioactive bound phenolics during in vitro gastrointestinal digestion and colonic fermentation boost the prebiotic effects of triticale insoluble dietary fiber

Phenolics in bound form extensively exist in cereal dietary fiber, especially insoluble fiber, while their release profile in gastrointestinal tract and contribution to the potential positive effects of dietary fiber in modulating gut microbiota still needs to be disclosed. In this work, the composition of bound phenolics (BPs) in triticale insoluble dietary fiber (TIDF) was studied, and in vitro gastrointestinal digestion as well as colonic fermentation were performed to investigate BPs liberation and their role in regulating intestinal flora of TIDF. It turned out that most BPs were unaccessible in digestion but partly released continuously during fermentation. 16 s rRNA sequencing demonstrated that TIDF possessed prebiotic effects by promoting anti-inflammatory while inhibiting proinflammatory bacteria alongside boosting SCFAs production and antioxidative BPs contributed a lot to these effects. Results indicated that TIDF held capabilities to regulate intestinal flora and BPs were important functional components to the health benefits of cereal dietary fiber.

Determination of fatty acid composition after saponification of common oil pharmaceutical excipients by supercritical fluid-evaporative light scattering method and its application in oil identification

Oils and fats are commonly used in the pharmaceutical industry as solvents, emulsifiers, wetting agents, and dispersants, and are an important category of pharmaceutical excipients. Fatty acids with unique compositions are important components of oil pharmaceutical excipients. The Chinese Pharmacopoeia provides clear descriptions of the fatty acid types and limits suitable for individual oil pharmaceutical excipient. An unqualified fatty acid composition or content may indicate adulteration or deterioration. The fatty acid composition, as a key indicator for the identification and adulteration evaluation of oil pharmaceutical excipients, can directly affect the quality and safety of oil pharmaceutical excipients and preparations. Gas chromatography is the most widely used technique for fatty acid analysis, but it generally requires derivatization, which affects quantitative accuracy. Supercritical fluid chromatography (SFC), an environmentally friendly technique with excellent separation capability, offers an efficient method for detecting fatty acids without derivatization. Unlike other chromatographic methods, SFC does not use nonvolatile solvents (e. g., water) as the mobile phase, rendering it compatible with an evaporative light-scattering detector (ELSD) for enhanced detection sensitivity. However, the fatty acids in oil pharmaceutical excipients exist in the free and bound forms, and the low content of free fatty acids in these oil pharmaceutical excipients not only poses challenges for their detection but also complicates the determination of characteristic fatty acid compositions and contents. Moreover, the compositions and ratios of fatty acids are influenced by environmental factors, leading to interconversion between their two forms. In this context, saponification provides a simpler and faster alternative to derivatization. Saponification degrades oils and fats by utilizing the reaction between esters and an alkaline solution, ultimately releasing the corresponding fatty acids. Because this method is more cost effective than derivatization, it is a suitable pretreatment method for the detection of fatty acids in oil pharmaceutical excipients using the SFC-ELSD approach. In this study, we employed SFC-ELSD to simultaneously determine six fatty acids, namely, myristic acid, palmitic acid, stearic acid, arachidic acid, docosanoic acid, and lignoceric acid, in oil pharmaceutical excipients. Saponification of the oil pharmaceutical excipients using sodium hydroxide methanol solution effectively avoided the bias in the determination of fatty acid species and contents caused by the interconversion of fatty acids and esters. The separation of the six fatty acids was achieved within 12 min, with good linearity within their respective mass concentration ranges. The limits of detection and quantification were 5-10 mg/L and 10-25 mg/L, respectively, and the spiked recoveries were 80.93%-111.66%. The method proved to be sensitive, reproducible, and stable, adequately meeting requirements for the analysis of fatty acids in oil pharmaceutical excipients. Finally, the analytical method was successfully applied to the determination of six fatty acids in five types of oil pharmaceutical excipients, namely, corn oil, soybean oil, coconut oil, olive oil, and peanut oil. It can be combined with principal component analysis to accurately differentiate different types of oil pharmaceutical excipients, providing technical support for the rapid identification and quality control of oil pharmaceutical excipients. Thus, the proposed method may potentially be applied to the analysis of complex systems adulterated with oil pharmaceutical excipients.