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16 pages, 3455 KiB  
Article
Genome-Wide Identification and Expression Analysis of CrRLK1-like Gene Family in Potatoes (Solanum tuberosum L.) and Its Role in PAMP-Triggered Immunity
by Yazhou Bao, Ru Zhao, Sixian Hu, Xiaoli Li, Like Wang, Ji Wang, Junbin Ji, Weiduo Wang, Changqing Zhu, Jiajia Chen, Ailing Ben, Jinfeng Peng and Tingli Liu
Genes 2025, 16(3), 308; https://doi.org/10.3390/genes16030308 - 4 Mar 2025
Viewed by 186
Abstract
Background: The Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) subfamily, a specialized group within receptor-like kinases (RLKs), was initially identified in C. roseus cell cultures. CrRLK1L plays an important role in the growth, development and stress response of plants. Although CrRLK1L genes have been [...] Read more.
Background: The Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) subfamily, a specialized group within receptor-like kinases (RLKs), was initially identified in C. roseus cell cultures. CrRLK1L plays an important role in the growth, development and stress response of plants. Although CrRLK1L genes have been characterized across multiple plant species, their biological and genetic functions in potatoes (Solanum tuberosum) remains poorly elucidated. Methods: a genome-wide investigation, phylogenetic analysis, chromosome localization, exon–intron structure, conserved motifs, stress-responsive cis-elements, tissue-specific expression patterns, and their effects on pathogen associated molecular patterns (PAMPs) induced reactive oxygen species (ROS) production were analyzed. Results: A total of 29 CrRLK1L genes were identified in the S. tuberosum genome, unevenly distributed across 10 chromosomes and divided into three groups. Tissue-specific expression analysis revealed seven genes highly expressed in all tissues, while CrRLK1L13 was specific to stamens and flowers. Under stress conditions (mannitol, salt, hormone, and heat), StCrRLK1L genes exhibited diverse expression patterns. Functional characterization in Nicotiana benthamiana identified seven ROS suppressors and four ROS enhancers, implicating their roles in PAMP-triggered immunity. Conclusions: This study provides valuable insights into the StCrRLK1L gene family, enhancing our understanding of its functions, particularly in plant innate immunity. Full article
(This article belongs to the Section Plant Genetics and Genomics)
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Figure 1
<p>Chromosomal locations of potato StCrRLK1L gene family members. Twenty-nine genes are mapped to 10 chromosomes based on Phytozome annotations.</p>
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<p>A phylogenetic tree of CrRLK1L-related proteins from <span class="html-italic">A. thaliana</span> (blue circles) and <span class="html-italic">S. tuberosum</span> (red circles). The tree was constructed using ClustalW alignment and MEGA X with 1000 bootstrap replicates.</p>
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<p>Structural and evolutionary analysis of StCrRLK1Ls. (<b>A</b>) Maximum-likelihood phylogenetic tree of 29 StCrRLK1Ls grouped into three subclasses. (<b>B</b>) Exon–intron architecture: green (UTRs), yellow (exons), gray line (introns). (<b>C</b>) Conserved motif distribution, colored by position in protein sequence.</p>
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<p><span class="html-italic">Cis</span>-element distribution in the StCrRLK1L promoters. Element positions are scaled relative to the translation start site (ATG).</p>
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<p>Heat map of StCrRLK1L expression across tissues in DM potatoes. Color intensity reflects log2-transformed FPKM values.</p>
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<p>Expression dynamics of the StCrRLK1L genes under stress conditions. The color scale was plotted using the log2 mean of FPKM of each gene.</p>
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<p>The Modulation of PAMP-triggered ROS by StCrRLK1Ls. The indicated constructs were transiently expressed by agrobacterium-mediated transient expression for 2 days and subjected to flg22-induced ROS examination (mean ± SD, <span class="html-italic">n</span> ≥ 8, and one-way ANOVA followed by Tukey’s post hoc test; different letters indicate significant difference at <span class="html-italic">p</span> &lt; 0.01). The protein expression is shown in <a href="#app1-genes-16-00308" class="html-app">Figure S1</a>.</p>
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15 pages, 4153 KiB  
Article
Highly Branched Poly(Adipic Anhydride-Co-Mannitol Adipate): Synthesis, Characterization, and Thermal Properties
by Mahir A. Jalal, Einas A. Abood, Zainab J. Sweah, Hadi S. Al-Lami, Alyaa Abdulhasan Abdulkarem and Haider Abdulelah
Polymers 2025, 17(5), 684; https://doi.org/10.3390/polym17050684 - 4 Mar 2025
Viewed by 128
Abstract
In this study, modification of poly(adipic anhydride) through branching its chains was carried out via melt condensation polymerization with D-mannitol. The percentage of mannitol was varied (3, 4, 5, 10, 15, and 20 Wt.%) and the resulting copolymers were purified and characterized by [...] Read more.
In this study, modification of poly(adipic anhydride) through branching its chains was carried out via melt condensation polymerization with D-mannitol. The percentage of mannitol was varied (3, 4, 5, 10, 15, and 20 Wt.%) and the resulting copolymers were purified and characterized by FT-IR and 13C-NMR. These analyses indicated that linear chains of poly(adipic anhydride) can react with strong nucleophiles and dissociate to produce highly branched poly(adipic anhydride-co-mannitol adipate) which confirms the validity of the proposed mechanism. The copolymer’s molecular weight characteristics have been also examined using GPC analysis. Thermal properties of copolymers were also investigated using TGA, DTG, and DCS analyses. TGA/DTG revealed that the thermal degradation of copolymers proceeds in multi-stage decomposition, whereas the shift and pattern change of the melting point peak of DSC curves can identify the weight percentage of mannitol for homogenous copolymers. Two non-isothermal models, the Flynn–Wall–Ozawa and Kissinger methods, have been also employed to analyze thermogravimetric data collected from the thermal decomposition of the copolymers and found that Flynn–Wall–Ozawa method provides better results with R2 correlation up to 99.3%. The activation energy in the region of Tmax was determined and found that an increase in mannitol contents in copolymer has a positive impact on its thermal stability. Full article
(This article belongs to the Section Polymer Analysis and Characterization)
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Graphical abstract

Graphical abstract
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<p>FT-IR of PAA and highly branched PAA-<span class="html-italic">co</span>-M copolymers.</p>
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<p><sup>13</sup>C NMR of (<b>a</b>) PAA and (<b>b</b>) PAA-<span class="html-italic">co</span>-M(10%). The blue scale represents to the chemical shift (ppm).</p>
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<p>TGA curves of PAA and PAA-<span class="html-italic">co</span>-M copolymers (<span class="html-italic">β</span> = 30 °C/min).</p>
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<p>DTG curves of PAA and PAA-<span class="html-italic">co</span>-M copolymers (<span class="html-italic">β</span> = 30 °C/min).</p>
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<p>DSC curves of copolymers at the region of their melting points.</p>
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<p>A plot of ln(<span class="html-italic">β</span>/<span class="html-italic">T<sub>p</sub></span><sup>2</sup>) versus the reciprocal of the peak temperature 1/<span class="html-italic">T<sub>p</sub></span> (Kissinger method) to estimate <span class="html-italic">E<sub>a</sub></span> for the thermal decomposition of copolymers.</p>
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<p>F-W-O plots for thermal degradation of PAA and PAA-<span class="html-italic">co</span>-M copolymers (<b>a</b>) first DTG peak and (<b>b</b>) second DTG peak.</p>
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<p>Steps of copolymer synthesis (<b>a</b>) polymerization of adipic acid and (<b>b</b>) chemical reaction of synthesis highly branched PAA-<span class="html-italic">co</span>-M.</p>
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16 pages, 3333 KiB  
Article
Freeze-Dried Liposomes as Carriers of Eugenia pyriformis Cambess Phytoactives for Cosmetic Applications
by Gabriela Alves Silva, Letícia Kakuda and Wanderley Pereira Oliveira
Processes 2025, 13(3), 693; https://doi.org/10.3390/pr13030693 - 28 Feb 2025
Viewed by 151
Abstract
The demand for phytoactives in cosmetics is growing due to their potential as safer and sustainable alternatives to synthetic compounds. The fruit pulp of Eugenia pyriformis Cambess (uvaia), a species native to the Atlantic Forest, is rich in phenolic compounds and ascorbic acid, [...] Read more.
The demand for phytoactives in cosmetics is growing due to their potential as safer and sustainable alternatives to synthetic compounds. The fruit pulp of Eugenia pyriformis Cambess (uvaia), a species native to the Atlantic Forest, is rich in phenolic compounds and ascorbic acid, with high antioxidant activity, making it a promising active ingredient for cosmetic applications, particularly in skin anti-aging formulations. This study aimed to extract bioactives from uvaia fruit, evaluate their antioxidant properties, and develop freeze-dried liposomes to enhance their stability and physicochemical characteristics. Uvaia pulp was freeze-dried and extracted via dynamic maceration using water (EX.AQ) and 70% ethanol (EX.ET). EX.ET exhibited the highest polyphenol content (16.44 ± 0.95 mg GAE/g dry basis) and antioxidant activity (IC50 = 122.09 ± 5.28 µg/mL). Liposomes containing EX.ET (F1-EX) and blank liposomes (F1-B) were prepared using an adapted ethanol injection method and freeze-dried with two cryoprotectant mixtures: A1 (trehalose, mannitol, and colloidal silicon dioxide) and P1 (whey protein and colloidal silicon dioxide). F1-EX liposomes had a larger particle size (232.50 ± 2.72 nm) than F1-B (143.90 ± 2.80 nm), with similar polydispersity (PdI < 0.30) and zeta potential (>30 mV). The encapsulation efficiency of F1-EX reached 58.8 ± 0.5%. Freeze-dried liposomes showed low water activity (0.068–0.340) and moisture content (2.71–3.58%), while cryoprotectant A1 resulted in a lower PdI (0.144 ± 0.039) and higher zeta potential (−39.00 ± 0.44). These results demonstrate that uvaia is a valuable source of bioactive substances, with encapsulation and drying technologies enhancing their stability and functionality, making them suitable for potential applications in the pharmaceutical and cosmetic sectors. Furthermore, this approach supports the sustainable use of natural resources and contributes to preserving Brazil’s biodiversity. Full article
(This article belongs to the Section Pharmaceutical Processes)
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Figure 1
<p>Voucher specimen of the uvaia tree (personal archive).</p>
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<p>Flowchart of the liposome production protocol.</p>
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<p>Results for the particle size (left, in blue) and polydispersity index (right, in red) for blank liposomes (F1-B) without Ultra-Turrax (ST), after Ultra-Turrax (PT), and based on the number of ultrasonication cycles (C).</p>
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<p>Results for the particle size (left, in blue) and polydispersity index (right, in red) for liposomes containing uvaia pulp hydroalcoholic extract (F1-EX) without Ultra-Turrax (ST), after Ultra-Turrax (PT), and based on the number of ultrasonication cycles (C).</p>
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<p>Encapsulation efficiency of liposomes containing uvaia pulp hydroalcoholic extract (F1-EX) following high-shear homogenization with Ultra-Turrax (PT), after one ultrasonication cycle (C1), and after two ultrasonication cycles (C2).</p>
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<p>Visual comparison of freeze-dried blank liposomes (F1-B) and liposomes containing uvaia pulp hydroalcoholic extract (F1-EX).</p>
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<p>Particle size of blank liposomes (F1-B) and liposomes containing uvaia pulp hydroalcoholic extract (F1-EX) with cryoprotectants before freeze-drying (NL) and after redispersion (R). * Indicates a statistically significant difference between redispersed liposomes (R) and non-freeze-dried liposomes (NL) (<span class="html-italic">p</span> ≤ 0.05).</p>
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<p>Zeta potential of blank liposomes (F1-B) and liposomes containing uvaia pulp hydroalcoholic extract (F1-EX) with cryoprotectants before freeze-drying (NL) and after redispersion (R). * Indicates a statistically significant difference between redispersed liposomes (R) and non-freeze-dried liposomes (NL) (<span class="html-italic">p</span> ≤ 0.05).</p>
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14 pages, 1534 KiB  
Article
Hippo Signaling Regulates High-NaCl-Induced Increase in RORγt+ Pro-Inflammatory Lymphocytes
by Bastian Lukas Zeeb, Saskia Weber-Stiehl, Celia Escudero-Hernández, Dominik N. Müller, Andras Maifeld, Felix Sommer, Roland Schmitt and Laura Katharina Sievers
Int. J. Mol. Sci. 2025, 26(5), 2143; https://doi.org/10.3390/ijms26052143 - 27 Feb 2025
Viewed by 268
Abstract
Arterial hypertension is a major health challenge worldwide. Lifestyle factors including dietary NaCl increase the risk of hypertension. Pathophysiologically, the activation of the renin–angiotensin–aldosterone system and vascular remodeling, as well as the increase in Th17 lymphocytes, contribute to increased blood pressure and end-organ [...] Read more.
Arterial hypertension is a major health challenge worldwide. Lifestyle factors including dietary NaCl increase the risk of hypertension. Pathophysiologically, the activation of the renin–angiotensin–aldosterone system and vascular remodeling, as well as the increase in Th17 lymphocytes, contribute to increased blood pressure and end-organ damage. To date, it is unknown whether NaCl, changed osmolarity, and/or angiotensin II directly induce Th17 differentiation, and, if so, which molecular pathways are involved. One major transcription factor inducing Th17 differentiation is RORγt. RORγt+ immune-cell subtypes increased in a mouse model of hypertension. In primary splenocytes, NaCl and mannitol but not angiotensin II increased the frequency of RORγt+ lymphocytes and IL-17 and IL-22 expression. NaCl and angiotensin II induced angiotensin II receptor expression. NaCl led to the inactivation of the Hippo pathway in lymphocytes and decreased phosphorylation of the transcription factor TAZ, leading to increased functionality as a transcriptional coregulator. Inhibition of TAZ by verteporfin blocked the NaCl-induced increase in RORγt+ lymphocytes. Taken together, we found that NaCl induced pro-inflammatory lymphocytes via the regulation of Hippo signaling. The results suggest the possible involvement of Hippo signaling in the pathophysiology of salt-sensitive hypertension, with the potential for therapeutic targeting by small-molecule approaches. Full article
(This article belongs to the Special Issue Cardiovascular Diseases: Aging and Other Risk Factors)
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<p>Immune cell subtypes were stable in an in vivo model of hypertension but show increased RORγt+ lymphocytes. (<b>a</b>) For the induction of chronic hypertension, mice were subjected to uninephrectomy, implantation of osmotic Ang-II mini pumps, and high dietary NaCl. Male and female mice were pooled, and the numbers varied by differences in survival. The control group consisted of 3 female and 4 male mice; in the Ang-II group, 2 were female and 4 male. (<b>b</b>) Spleens were lysed and analyzed via flow cytometry: T cells (CD3+), B cells (CD45+, CD3-, CD19+), cytotoxic T cells (Tc; CD3+ gdTCR- CD4- CD8+), T-helper cells (Th; CD3+ γδTCR- CD4+ CD8-), Treg (CD3+ γδTCR- CD4+ CD8- FOXP3+), conventional T-helper cells (Tconv.; CD3+ γδTCR- CD4+ CD8- FOXP3-), and innate lymphoid cells (ILCs; CD45+ CD3-,TBET+ RORγt+ GATA3+). Each dot denotes one animal. Statistical test: unpaired, two-tailed <span class="html-italic">t</span>-test (<span class="html-italic">p</span>-value given).</p>
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<p>NaCl induced CD3e+CD4+RORγt+ lymphocytes. Flow cytometry of primary cultured splenocytes that were treated for 24 h with NaCl, angiotensin II (Ang II), and mannitol and stained for the main lymphocyte marker proteins: RORγt (e.g., Th17 cells), FoxP3 (Treg), Tbet (Th1), and Gata3 (Th2). Each dot denotes data from one spleen culture. n = 31; statistical test: one-way ANOVA; ****, <span class="html-italic">p</span> &lt; 0.0001; *** <span class="html-italic">p</span> &lt; 0.001 and * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Hippo signaling components TAZ and CTGF were expressed higher in Th17 than naive and Treg: (<b>a</b>) regularized logarithmic gene expression of selected genes in murine T-lymphocyte subtypes; (<b>b</b>) ratio of the regularized logarithmic expression of all genes between Th17/Treg and Th17/naive T cells. Each dot depicts one gene. The <span class="html-italic">Wwtr1</span> gene, which codes for the TAZ protein, and the TAZ target gene <span class="html-italic">Ctgf</span> are marked in red (<span class="html-italic">Wwtr1</span>: 2.36/2.26; <span class="html-italic">C</span>tgf: 3.77/3.69; all data provided in <a href="#app1-ijms-26-02143" class="html-app">Table S1</a>) [<a href="#B7-ijms-26-02143" class="html-bibr">7</a>].</p>
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<p>NaCl induced proinflammatory cytokines. mRNA expression analyses via qPCR of human immortalized lymphocytes cultured under the indicated conditions for 24 h; n = 4. statistical test: one-way ANOVA; *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.005, and * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>NaCl induced phosphorylation and increased nuclear localization of TAZ in lymphocytes. (<b>a</b>) Semiquantitative Western blots of protein expression from human immortalized lymphocytes. n = 6; statistical test: one-way ANOVA; *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.005 and * <span class="html-italic">p</span> &lt; 0.05. (<b>b</b>) Representative immunofluorescence staining of TAZ and YAP (green) in primary cultured splenic lymphocytes treated with NaCl or mannitol for 24 h. Cytoskeleton was contrasted (red) and nuclei were stained with DAPI (blue). The scale bar denotes 20 µm. (<b>c</b>) The fluorescence intensity ratios of YAP/TAZ in the nuclei and cytoplasm of 10 arbitrarily chosen cells were calculated. n = 10; statistical test: one-way ANOVA; ** <span class="html-italic">p</span> &lt; 0.005 and * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Inhibition of TAZ by verteporfin blocked the NaCl-induced increase in CD3e+CD4+RORγt+ lymphocytes. Flow cytometry of primary cultured splenocytes treated with NaCl or mannitol for 24 h in the presence or absence of verteporfin, an inhibitor of TAZ, and stained for the main lymphocyte marker proteins RORγt (e.g., Th17 cells), FoxP3 (Treg), Tbet (Th1), and Gata3 (Th2). n = 31, for the control; n = 17, for verteporfin. Statistical test: one-way ANOVA; **** <span class="html-italic">p</span> &lt; 0.0001 and *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Hippo signaling regulates high-NaCl-induced increases in RORγt+ pro-inflammatory lymphocytes. Schematic overview of the results.</p>
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15 pages, 3682 KiB  
Article
Paracoccus broussonetiae subsp. drimophilus subsp. nov., a Novel Subspecies Salt-Tolerant Endophytic Bacterium from Maize Root in Hunan
by Xue Li, Chi Zhou, Ming Li, Qingzhuang Zhang, Lei Su and Xin Li
Life 2025, 15(3), 354; https://doi.org/10.3390/life15030354 - 24 Feb 2025
Viewed by 215
Abstract
In an investigation exploring endophytic microbiota from agricultural crops, an aerobic, non-motile, Gram-negative, coccobacillus-shaped bacterial isolate, designated as strain NGMCC 1.201697T, was isolated from maize roots in Hunan Province, China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain [...] Read more.
In an investigation exploring endophytic microbiota from agricultural crops, an aerobic, non-motile, Gram-negative, coccobacillus-shaped bacterial isolate, designated as strain NGMCC 1.201697T, was isolated from maize roots in Hunan Province, China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NGMCC 1.201697T belonged to the genus Paracoccus, showing the highest sequence similarity to Paracoccus broussonetiae CPCC 101403T (99.86%). The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) were 98.57% and 87.90% between the novel isolate and its closest phylogenetic relative. However, phenotypic characterization further differentiated the isolate from P. broussonetiae CPCC 101403T. The isolate showed enhanced environmental tolerance adaptability (growth in 0–8% NaCl and 4–37 °C), unique enzymatic activities (esterase C4, β-glucosidase, L-proline arylamidase, and β-galactosidase), and expanded metabolic capabilities (D-mannitol, D-cellobiose, saccharose, and so on). The major polar lipids consisted of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), two unidentified glycolipids (GLs) and four unidentified phospholipids (PLs). The predominant respiratory quinone was ubiquinone-10, and the major fatty acid was summed feature 8 (C18:1 ω7c, 69.42%). The DNA G + C content was 64.49 mol%. Based on results of these analyses, strain NGMCC 1.201697T represents a novel subspecies of Paracoccus broussonetiae, for which the name Paracoccus broussonetiae subsp. drimophilus subsp. nov. is proposed. The type-strain is NGMCC 1.201697T (=CGMCC 1.61958T =JCM 37104T). Full article
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Figure 1
<p>Transmission electron micrograph (TEM) image of strain NGMCC 1.201697<sup>T</sup> during the exponential stage of growth. Bar, 1 μm and 2 μm.</p>
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<p>Maximum likelihood (ML) phylogenetic tree based on 16S rRNA gene evolutionary distances among strain NGMCC 1.201697<sup>T</sup> and members of the genus <span class="html-italic">Paracoccus</span>. Bootstrap values (expressed as percentages of 1000 replications) above 50% are shown at branch points. Bar 0.01 substitutions per nucleotide position. <span class="html-italic">Rhodobacter capsulatus</span> ATCC 11166<sup>T</sup> (D16428) was used as an outgroup.</p>
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<p>Phylogenomic tree showing the position of strain NGMCC 1.201697<sup>T</sup> based on bac120 marker set by maximum likelihood inference. Bootstrap values above 50% are shown at the nodes. Bar, 0.01 substitutions per nucleotide position. <span class="html-italic">Rhodobacter capsulatus</span> ATCC 11166<sup>T</sup> (QKZO01000000) was used as an outgroup.</p>
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<p>Analysis of the KEGG function classification (<b>A</b>), and analysis of the COG function classification (<b>B</b>) based on the genomes of stain NGMCC 1.201697<sup>T</sup>.</p>
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<p>Polar lipids profile of strain NGMCC 1.201697<sup>T</sup>. The plates in <a href="#life-15-00354-f005" class="html-fig">Figure 5</a> (<b>A</b>–<b>D</b>) are sprayed with molybdophosphoric acid, ninhydrin, α-naphthol and molybdenum blue staining agent to show all polar lipids present, respectively. DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; GL1–2, two unidentified glycolipids; PL1–4, unidentified phospholipid.</p>
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21 pages, 3103 KiB  
Article
Sustainable Recovery of Phlorotannins from Durvillaea incurvata: Integrated Extraction and Purification with Advanced Characterization
by Pamela Raquel Rivera-Tovar, Gabriela Contreras-Contreras, Paulina Isabel Rivas-Reyes, Jara Pérez-Jiménez, Maximiliano Martínez-Cifuentes, José Ricardo Pérez-Correa and María Salomé Mariotti-Celis
Antioxidants 2025, 14(3), 250; https://doi.org/10.3390/antiox14030250 - 21 Feb 2025
Viewed by 189
Abstract
The rising demand for bioactive compounds from marine resources highlights the need for sustainable separation technologies. This study introduces an integrated process combining ultrasound-assisted extraction (USAE) and resin purification (RP) to isolate phlorotannins from Durvillaea incurvata, a brown seaweed with significant biomedical [...] Read more.
The rising demand for bioactive compounds from marine resources highlights the need for sustainable separation technologies. This study introduces an integrated process combining ultrasound-assisted extraction (USAE) and resin purification (RP) to isolate phlorotannins from Durvillaea incurvata, a brown seaweed with significant biomedical potential. Using a 32.5% ethanol–water solvent system for USAE followed by RP on Diaion HP-20 resin, phlorotannins were enriched 2.4-fold, with simultaneous removal of interfering compounds such as mannitol (~100%), which was demonstrated by FTIR and HPLC-IR analysis. Advanced characterization using UHPLC-QToF-MS/MS identified five novel phlorotannins with polymerization degrees of 3 to 8 phloroglucinol units in both USAE extracts and post-RP. Mass balance based on spectrophotometric measurements indicated a purification factor of ~2, confirming process effectiveness. RP streams showed distinct phlorotannin profiles, with one phlorotannin exceeding 70% relative abundance. However, MS/MS results showed significantly lower recoveries than spectrophotometric data, revealing a novel insight into RP purification. These findings highlight the critical role of comprehensive chemical characterization in optimizing sustainable phlorotannin extraction from seaweed. They propose a framework for scalable, eco-efficient technologies for achieving high-purity phlorotannin recovery. This approach facilitates the development of phlorotannin-based applications in the nutraceutical and pharmaceutical industries. Full article
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Figure 1
<p>(<b>A</b>) TPC and TPhC, and (<b>B</b>) antioxidant capacity (DPPH and ORAC assays) of RE, CE, and RP streams (CS, WS, and ES) from <span class="html-italic">Durvillaea incurvata</span> frond. <sup>(A–D; a–d)</sup> Values that do not share a letter are significantly different.</p>
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<p>(<b>a</b>) FTIR spectra of mannitol, phloroglucinol, CE, and RP streams. (<b>b</b>) Characteristic bands of each FTIR spectrum associated with specific types of vibrations.</p>
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<p>Proposed structures of the five identified phlorotannins in the extracts of <span class="html-italic">Durvillaea incurvata</span> frond.</p>
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<p>Distribution of the five proposed phlorotannins identified in the process streams (CE, CS, WS, and ES) from <span class="html-italic">Durvillaea incurvata</span> frond.</p>
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<p>Relative mass balance of the integrated process of USAR-RP from <span class="html-italic">Durvillaea incurvata</span> frond. Relative abundances correspond to the ratio of the relative area of phlorotannin and ExS of the stream.</p>
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<p>Percentage distribution of ExS, TP, TPh, Mannitol, and specific phlorotannins of CE in the RP streams.</p>
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18 pages, 4747 KiB  
Article
Evaluation of Permeability, Safety, and Stability of Nanosized Ketoprofen Co-Spray-Dried with Mannitol for Carrier-Free Pulmonary Systems
by Heba Banat, Ilona Gróf, Mária A. Deli, Rita Ambrus and Ildikó Csóka
Appl. Sci. 2025, 15(3), 1547; https://doi.org/10.3390/app15031547 - 3 Feb 2025
Viewed by 633
Abstract
Pulmonary drug delivery presents a promising approach for managing respiratory diseases, enabling localized drug deposition and minimizing systemic side effects. Building upon previous research, this study investigates the cytotoxicity, permeability, and stability of a novel carrier-free dry powder inhaler (DPI) formulation comprising nanosized [...] Read more.
Pulmonary drug delivery presents a promising approach for managing respiratory diseases, enabling localized drug deposition and minimizing systemic side effects. Building upon previous research, this study investigates the cytotoxicity, permeability, and stability of a novel carrier-free dry powder inhaler (DPI) formulation comprising nanosized ketoprofen (KTP) and mannitol (MNT). The formulation was prepared using wet media milling to produce KTP-nanosuspensions, followed by spray drying to achieve combined powders suitable for inhalation. Cell viability and permeability were conducted in both alveolar (A549) and bronchial (CFBE) models. Stability was assessed after storage in hydroxypropyl methylcellulose (HPMC) capsules under stress conditions (40 °C, 75% RH), as per ICH guidelines. KTP showed good penetration through both models, with lower permeability through the CFBE barrier. The MNT-containing sample (F1) increased permeability by 1.4-fold in A549. All formulations had no effect on cell barrier integrity or viability after the impedance test, confirming their safety. During stability assessment, the particle size remained consistent, and the partially amorphous state of KTP was retained over time. However, moisture absorption induced surface roughening and partial agglomeration, leading to reduced fine particle fraction (FPF) and emitted fraction (EF). Despite these changes, the mass median aerodynamic diameter (MMAD) remained stable, confirming the formulation’s continued applicability for pulmonary delivery. Future research should focus on optimizing excipient content, alternative capsule materials, and storage conditions to mitigate moisture-related issues. Hence, the findings demonstrate that the developed ketoprofen–mannitol DPI retains its quality and performance characteristics over an extended period, making it a viable option for pulmonary drug delivery. Full article
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<p>Cell viability of (<b>A</b>) A549 alveolar and (<b>B</b>) CFBE bronchial epithelial cells after 1-h treatments with KTP raw and the developed F0, F1 samples measured by impedance. Values are presented as means ± SD, <span class="html-italic">n</span> = 5–6. Statistical analysis: ANOVA followed by Dunett’s test. *** <span class="html-italic">p</span> &lt; 0.001 compared to the control group. C, control; KTP, ketoprofen raw; TX-100, Triton X-100 detergent.</p>
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<p>Permeability of KTP raw, F0, and F1 DPIs (50 μg/mL KTP concentration in the donor compartment) across the co-culture model of (<b>A</b>) alveolar epithelial cells and (<b>B</b>) bronchial epithelial cells after 30- and 60-min assay time. Values are presented as means ± SD, <span class="html-italic">n</span> = 4. Statistical analysis: ANOVA followed by Dunett’s test. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Evaluation of the tightness of the A549 alveolar and the CFBE bronchial epithelial co-culture models after the KTP raw, F0, and F1 DPIs permeability experiment by (<b>A</b>,<b>C</b>) transepithelial electrical resistance (TEER) measurement and the (<b>B</b>,<b>D</b>) permeability of fluorescein and albumin marker molecules. Values are presented as means ± SD, <span class="html-italic">n</span> = 4. Statistical analysis: ANOVA followed by Dunett’s test.</p>
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<p>Validation of barrier integrity in A549 alveolar epithelial and CFBE bronchial epithelial cell layers after permeability assays with KTP raw, F0, and F1. Immunostaining for (<b>A</b>) β-catenin and (<b>B</b>) zonula occludens protein-1. Cyan: cell nuclei; red: junctional proteins; scale bar: 20 μm. C, control; KTP, ketoprofen raw.</p>
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<p>Laser diffraction results of particle size (D[0.5] (µm)) and particle size distribution (Span) for sample F1 (freshly prepared; F1—0m, and after one month; F1—1m and 3 months; F1—3m of storage). Results are presented as means ± SD, <span class="html-italic">n</span> = 3., **** <span class="html-italic">p</span> &lt; 0.0001 significantly different.</p>
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<p>SEM images of F1 sample at (<b>a</b>) 0 months, (<b>b</b>) 1 month, and (<b>c</b>) 3 months.</p>
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<p>DSC thermal analysis of (<b>A</b>) raw ketoprofen (KTP) and the physical mixture (PM), and (<b>B</b>) sample F1, analyzed as a freshly prepared sample (F1—0m) and after 1 (F1—1m) and 3 (F1—3m) months of storage.</p>
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<p>DSC thermal analysis of (<b>A</b>) raw ketoprofen (KTP) and the physical mixture (PM), and (<b>B</b>) sample F1, analyzed as a freshly prepared sample (F1—0m) and after 1 (F1—1m) and 3 (F1—3m) months of storage.</p>
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<p>XRPD structural analysis of (<b>A</b>) raw ketoprofen (KTP) and the physical mixture (PM), and (<b>B</b>) sample F1, analyzed as a freshly prepared sample (F1—0m) and after 1 (F1—1m) and 3 (F1—3m) months of storage. The circles highlight the characteristic peaks of KTP in the raw drug, physical mixture, as well as fresh and stored formulations.</p>
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<p>In vitro aerodynamic characteristics (MMAD, FPF, and EF) of sample F1 were analyzed as a freshly prepared sample (F1—0m), and after 1 (F1—1m) and 3 (F1—3m) months of storage, at a flow rate of 60 L/min. Data are presented as means ± SD (<span class="html-italic">n</span> = 3 independent measurements). Statistical significance is indicated, **** <span class="html-italic">p</span> &lt; 0.0001 significantly different.</p>
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<p>In vitro aerodynamic distribution of the F1 sample was analyzed as a freshly prepared sample (F1—0m), and after 1 (F1—1m) and 3 (F1—3m) months of storage, at a flow rate of 60 L/min. Data are presented as means ± SD (<span class="html-italic">n</span> = 3 independent measurements). Statistical significance is indicated as follows: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p>
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<p>In vitro release study of raw KTP, and spray-dried sample (F1) was analyzed as a freshly prepared sample (F1—0m) and after 1 (F1—1m) and 3 (F1—3m) months of storage, in simulated lung media (SLM). Results are expressed as mean ± SD (<span class="html-italic">n</span> = 3 independent measurements).</p>
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17 pages, 5094 KiB  
Article
Extrusion-Based 3D Printing of Pharmaceuticals—Evaluating Polymer (Sodium Alginate, HPC, HPMC)-Based Ink’s Suitability by Investigating Rheology
by Farzana Khan Rony, Georgia Kimbell, Toby R. Serrano, Destinee Clay, Shamsuddin Ilias and Mohammad A. Azad
Micromachines 2025, 16(2), 163; https://doi.org/10.3390/mi16020163 - 30 Jan 2025
Cited by 1 | Viewed by 776
Abstract
Three-dimensional printing is promising in the pharmaceutical industry for personalized medicine, on-demand production, tailored drug loading, etc. Pressure-assisted microsyringe (PAM) printing is popular due to its low cost, simple operation, and compatibility with heat-sensitive drugs but is limited by ink formulations lacking the [...] Read more.
Three-dimensional printing is promising in the pharmaceutical industry for personalized medicine, on-demand production, tailored drug loading, etc. Pressure-assisted microsyringe (PAM) printing is popular due to its low cost, simple operation, and compatibility with heat-sensitive drugs but is limited by ink formulations lacking the essential characteristics, impacting their performance. This study evaluates inks based on sodium alginate (SA), hydroxypropyl cellulose (HPC H), and hydroxypropyl methylcellulose (HPMC K100 and K4) for PAM 3D printing by analyzing their rheology. The formulations included the model drug Fenofibrate, functional excipients (e.g., mannitol, polyethylene glycol, etc.), and water or water–ethanol mixtures. Pills and thin films as an oral dosage were printed using a 410 μm nozzle, a 10 mm/s speed, a 50% infill density, and a 60 kPa pressure. Among the various formulated inks, only the ink containing 0.8% SA achieved successful prints with the desired shape fidelity, linked to its rheological properties, which were assessed using flow, amplitude sweep, and thixotropy tests. This study concludes that (i) an ink’s rheological properties—viscosity, shear thinning, viscoelasticity, modulus, flow point, recovery, etc.—have to be considered to determine whether it will print well; (ii) printability is independent of the dosage form; and (iii) the optimal inks are viscoelastic solids with specific rheological traits. This research provides insights for developing polymer-based inks for effective PAM 3D printing in pharmaceuticals. Full article
(This article belongs to the Special Issue Future Prospects of Additive Manufacturing)
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<p>Schematic presentation of the steps involved in the pressure-assisted microsyringe (PAM)-type extrusion-based 3D printing process.</p>
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<p>Molecular structure of sodium alginate (SA), hydroxypropyl cellulose (HPC), and hydroxypropyl methylcellulose (HPMC) [<a href="#B34-micromachines-16-00163" class="html-bibr">34</a>,<a href="#B35-micromachines-16-00163" class="html-bibr">35</a>].</p>
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<p>Design of pill and thin film used for 3D printing.</p>
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<p>Schematic presentation of the 3D printing of pills and thin films.</p>
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<p>Three-dimensional printing outcomes using various polymer-based inks. The printing conditions were a 50% infill density, a 10 mm/s print speed, and a 60 kPa printing pressure.</p>
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<p>Viscosity curves of (<b>a</b>) sodium alginate-, (<b>b</b>) HPC H-, and (<b>c</b>,<b>d</b>) HPMC K100/K4-based ink. The tests were performed at 25 °C, a 1 Hz frequency, and a 0.1 to 1000 1/s shear rate.</p>
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<p>Amplitude sweep curves (modulus vs. shear strain) of (<b>a</b>) sodium alginate-, (<b>b</b>) HPC H-, and (<b>c</b>,<b>d</b>) HPMC K100/K4-based ink. The tests were performed at 25 °C, a 1 Hz frequency, and a 0.001 to 1000%. shear strain.</p>
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<p>Thixotropy profiles of (<b>a</b>) sodium alginate-, (<b>b</b>) HPC H-, (<b>c</b>,<b>d</b>) HPMC K100/K4-based ink.</p>
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27 pages, 5607 KiB  
Article
Experimental Investigation into the Design, Optimization, Toxicity, and Anti-Viral Efficacy of Proliposomes Loaded with Ivermectin Against Infectious Bronchitis Virus Using an Embryonated Chicken Egg Model
by Mohammad H. Alyami, Hamad S. Alyami, Asmaa M. Abdo, Shereen A. Sabry, Shimaa M. G. Mansour, Hanan M. El-Nahas and Margrit M. Ayoub
Pharmaceutics 2025, 17(2), 165; https://doi.org/10.3390/pharmaceutics17020165 - 25 Jan 2025
Viewed by 836
Abstract
Background: Infectious bronchitis virus (IBV) causes a significant illness in birds, making it a leading source of financial loss in the poultry business. The objective of this study was to assess the effectiveness of proliposomes (PLs) containing ivermectin (IVM) against IBV using [...] Read more.
Background: Infectious bronchitis virus (IBV) causes a significant illness in birds, making it a leading source of financial loss in the poultry business. The objective of this study was to assess the effectiveness of proliposomes (PLs) containing ivermectin (IVM) against IBV using embryonated chicken eggs (ECEs). Methods: A three-factor, two-level (23) full factorial design was employed; carrier/lipid phase ratio (A), stearyl glycyrrhetinate amount (B), and phospholipid type (C) were studied as independent variables, while product yield (PY), entrapment efficiency (EE), particle size (PS), polydispersity index (PDI), zeta potential (ZP), and cumulative percentage of drug released after 6 h (Q6h) were characterized. The selected formulations (PL2 and PL6) were subjected to further characterizations, including IVM toxicity and anti-viral activity. Results: The PY% ranged from 88.6 ± 2.19% to 98.8 ± 0.45%, EE% was from 71.8 ± 2.01% to 96.1 ± 0.51%, PS was from 330.1 ± 55.65 nm to 1801.6 ± 45.61 nm, PDI was from 0.205 ± 0.06 to 0.603 ± 0.03, ZP was from −18.2 ± 0.60 mV to −50.1 ± 1.80 mV, and Q6h was from 80.95 ± 1.36% to 88.79 ± 2.03%. IVM-loaded PLs had lower toxicity in ECEs than pure IVM; the mortality rate was substantially reduced in IBV-infected ECEs injected with PL2 rather than pure IVM. As further evidence of IVM’s anti-viral action against IBV, quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the PL2-treated group exhibited further reduction in IBV’s copies in comparison with the pure IVM-treated group. Conclusions: PLs loaded with IVM are an innovative and potentially effective way to inhibit IBV. Full article
(This article belongs to the Section Drug Delivery and Controlled Release)
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<p>Three-dimensional response surface plots presenting the influence of the independent factors on (<b>a</b>) PY%, (<b>b</b>) EE%, (<b>c</b>) PS, (<b>d</b>) PDI, (<b>e</b>) ZP, and (<b>f</b>) Q6h.</p>
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<p>Influence of the independent factors on Y<sub>1</sub> response (PY%). (<b>a</b>) Effect of A at medium level of B and C. (<b>b</b>) Effect of B at medium level of A and C. (<b>c</b>) Effect of C at medium level of A and B.</p>
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<p>Influence of the independent factors on Y<sub>2</sub> response (EE%). (<b>a</b>) Effect of A at medium level of B and C. (<b>b</b>) Effect of B at medium level of A and C. (<b>c</b>) Effect of C at medium level of A and B.</p>
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<p>Influence of the independent factors on Y<sub>3</sub> response (PS). (<b>a</b>) Effect of A at medium level of B and C. (<b>b</b>) Effect of B at medium level of A and C. (<b>c</b>) Effect of C at medium level of A and B.</p>
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<p>Influence of the independent factors on Y<sub>5</sub> response (ZP). (<b>a</b>) Effect of A at medium level of B and C. (<b>b</b>) Effect of B at medium level of A and C. (<b>c</b>) Effect of C at medium level of A and B.</p>
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<p>Scanning electron micrographs (<b>a</b>) mannitol, (<b>b</b>) PL2, (<b>c</b>) PL6 at 5000×, and (<b>d</b>) photomicrograph of liposomal dispersion which formed upon hydration of DPPC based PLs.</p>
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<p>(<b>a</b>,<b>b</b>) DSC thermograms of pure IVM, SG, cholesterol, SPC, mannitol, selected formulations (PL2 and PL6), and the blank formulation.</p>
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<p>FTIR spectra of pure IVM, SG, SPC, cholesterol, mannitol, the selected formulations (PL2 and PL6), and the blank formulation.</p>
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<p>Outline of stability studies for selected formulations for 1, 2, and 3 months at 4 ± 1 °C and 25 ± 1 °C in terms of (<b>a</b>) EE% of PL2, (<b>b</b>) EE% of PL6, (<b>c</b>) PS of PL2, (<b>d</b>) PS of PL6, (<b>e</b>) ZP of PL2, and (<b>f</b>) ZP of PL6. * Significant (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Macroscopic lesions of embryos in negative control and drug-inoculated groups. (<b>a</b>) Negative control embryo showing normal appearance of liver. (<b>b</b>) Embryo inoculated with 100 µM of PL6 showing pale liver with hemorrhagic area. (<b>c</b>) Embryo inoculated with 400 µM of PL2 showing severely congested liver. (<b>d</b>) Embryo inoculated with 400 µM of pure IVM showing congested liver.</p>
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<p>Influence of pure IVM, PL2, and PL6 in the percentage of mortality of IBV-infected ECEs. The positive control group included 10<sup>3</sup> EID50 per 100 µL of IB MA5 virus. Data are the result of two independent experiments performed in duplicate. Statistical significance was assessed by two-way analysis of variance (ANOVA) with Tukey post hoc test. Values are expressed as mean ± standard error of mean. * <span class="html-italic">p</span> &lt; 0.05 in comparison to negative control group; # <span class="html-italic">p</span> &lt; 0.05 in comparison to positive control group.</p>
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<p>(<b>A</b>) Macroscopic visualization of embryos in negative control, IBV-infected and virus-treated inoculated ECEs. [(<b>a</b>) Uninoculated control embryo. (<b>b</b>) IBV-infected embryo treated with 200 µM of PL2. (<b>c</b>) Curling and dwarfing in embryo inoculated with IBV only]. (<b>B</b>) The lengths of embryos in negative control, IBV-infected and virus-treated inoculated ECEs. The positive control group included 10<sup>3</sup> EID50 per 100 µL of IB MA5 virus. Data are the result of two independent experiments performed in duplicate. Statistical significance was assessed by two-way analysis of variance (ANOVA) with Tukey post hoc test. Values are expressed as mean ± standard error of mean. *** <span class="html-italic">p</span> &lt; 0.001 in comparison to negative control group and ### <span class="html-italic">p</span> &lt; 0.001 in comparison to positive control group.</p>
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<p>Influence of 200 µM of pure IVM, PL2, and PL6 in the viral load of IBV-infected ECEs 72 h post inoculation. The positive control group included 10<sup>3</sup> EID50 per 100 µL of IB MA5 virus. Data are the result of two independent measurements. Statistical significance was assessed by one-way analysis of variance (ANOVA) with Tukey post hoc test. Values are expressed as mean ± standard error of mean. *** <span class="html-italic">p</span> &lt; 0.001; ** <span class="html-italic">p</span> &lt; 0.01 in comparison to positive control group.</p>
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20 pages, 650 KiB  
Review
Fluid and Electrolyte Disorders in Traumatic Brain Injury: Clinical Implications and Management Strategies
by Vivie Tran, Jackeline Flores, Meagan Sheldon, Camilo Pena and Kenneth Nugent
J. Clin. Med. 2025, 14(3), 756; https://doi.org/10.3390/jcm14030756 - 24 Jan 2025
Viewed by 1121
Abstract
Traumatic brain injuries (TBIs) cause direct central nervous system injury. The presentation depends on the location, the type, and the severity of the injury. Additional injury may develop secondary to compression, the disruption of cerebral perfusion, and changes in sodium levels, resulting in [...] Read more.
Traumatic brain injuries (TBIs) cause direct central nervous system injury. The presentation depends on the location, the type, and the severity of the injury. Additional injury may develop secondary to compression, the disruption of cerebral perfusion, and changes in sodium levels, resulting in either cellular edema or dehydration. Plasma osmolality (Posm) is a critical parameter influenced by solute concentrations, including sodium, glucose, and urea, and is a relevant concern when considering sodium levels in these patients. While Posm can be calculated using a standard formula, direct measurements via osmometry offer better accuracy. It is essential to differentiate between osmolality and tonicity; the latter refers specifically to effective solutes that drive water movement in the extracellular fluid. Sodium and its anions are effective solutes, whereas urea and glucose have variable effects due to their permeability and insulin dependence. Following TBI, the dysregulation of osmoregulation may occur and affect neurological outcomes. Osmoreceptors in the brain regulate arginine vasopressin secretion in response to changes in effective solute concentrations, with sodium chloride and mannitol being potent stimuli. The regulation of plasma osmolality, typically maintained within ±5% of the 280–295 mOsm/kg H2O range, is crucial for homeostasis and relies on antidiuresis and thirst mechanisms. This review narrative underscores the complexities of osmoregulation in the context of TBIs and their clinical implications, particularly concerning the development of conditions such as diabetes insipidus, the syndrome of inappropriate antidiuretic hormone secretion, and abnormal thirst. Full article
(This article belongs to the Section Brain Injury)
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<p>Schematic representation of blood-brain barrier (BBB). Panel <b>A</b> shows in physiologic conditions the different components including astrocyte end foot processes, pericytes, Aquaporin-4 (AQP4) channels, and endothelial cells. Panel <b>B</b> shows part of the findings when disruption of the BBB occurs with traumatic brain injury leading to development of cerebral edema, passage of substances presumably plasma, platelets, red and white cells, fibrinogen, immunoglobulins, among others, and the role of the microglia activation in a pro-inflammatory state. Created by JF December 2024.</p>
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15 pages, 1323 KiB  
Article
Functional Kimchi Beverage Enhanced with γ-Aminobutyric Acid (GABA) Through Serial Co-Fermentation Using Leuconostoc citreum S5 and Lactiplantibacillus plantarum KS2020
by Min-Jeong Kwon, Ji-Eun Kim and Sam-Pin Lee
Fermentation 2025, 11(1), 44; https://doi.org/10.3390/fermentation11010044 - 19 Jan 2025
Viewed by 956
Abstract
A plant-based beverage enhanced with GABA was developed through serial co-fermentation using Leuconostoc citreum S5 and Lactiplantibacillus plantarum KS2020. The first lactic acid fermentation was performed by Leu. citreum S5 with a vegetable mixture consisting of sliced radish, ginger, garlic, red pepper, bell [...] Read more.
A plant-based beverage enhanced with GABA was developed through serial co-fermentation using Leuconostoc citreum S5 and Lactiplantibacillus plantarum KS2020. The first lactic acid fermentation was performed by Leu. citreum S5 with a vegetable mixture consisting of sliced radish, ginger, garlic, red pepper, bell pepper, and sucrose. The viable cell count of Leu. citreum S5 increased to 9.11–9.42 log CFU/mL with higher sucrose contents, indicating the highest value of 9.42 log CFU/mL at 5% sucrose on day 1. Mannitol and dextran production levels in the first fermented vegetable mixture were 6.66–14.54 mg/mL and 0.44–2.26%, respectively. A higher sucrose content produced more dextran, resulting in a concomitant increase in viscosity of 49.4 mPa·s. The second co-fermentation for the kimchi beverage base was performed by Lb. plantarum KS2020 for 5 days, resulting in 8.22–9.60 log CFU/mL. The pH of the co-fermented kimchi beverage base increased to 6.19–9.57 with an increasing monosodium glutamate (MSG) content (3–7%), while titratable acidity significantly decreased to 0.0–0.8%. The final co-fermented kimchi beverage base was enriched with 2.6% GABA. Consequently, a GABA kimchi beverage base with probiotics, a red pigment, and a pleasant flavor was developed using only vegetable ingredients by serial co-fermentation using lactic acid bacteria. Full article
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<p>Viable cell count of the kimchi beverage base during co-fermentation. Changes in <span class="html-italic">Leu. citreum</span> S5 and <span class="html-italic">Lb. plantarum</span> KS2020 according to sucrose content. S0, sucrose-free; sucrose content added: S1 (1% sucrose); S3 (3% sucrose); S5 (5% sucrose). The results present the mean ± standard deviation (<span class="html-italic">n</span> = 3); Statistically significant differences were observed between the groups (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Free sugar content in the kimchi beverage base during co-fermentation. Changes in sucrose, fructose, glucose, and mannitol content according to sucrose content. S0, sucrose-free; sucrose content added: S1 (1% sucrose); S3 (3% sucrose); S5 (5% sucrose). The results present the mean ± standard deviation (<span class="html-italic">n</span> = 3); Statistically significant differences were observed between the groups (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Dextran content and apparent viscosity in the vegetable mixture fermented by <span class="html-italic">Leu. citreum</span> S5. (<b>A</b>) Differences in soluble and insoluble dextran content depending on sucrose content. (<b>B</b>) Changes depending on sucrose content and rotational speed; viscosity = dial reading × factor (speed 6, 10; 12, 5; 30, 2; 60, 1). S0, sucrose-free; sucrose content added: S1 (1% sucrose); S3 (3% sucrose); S5 (5% sucrose). The results present the mean ± standard deviation (<span class="html-italic">n</span> ≥ 3); Different letters (<sup>A–D</sup>) and (<sup>a–d</sup>) indicate statistically significant differences between the conditions and rotational speed, respectively, (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>pH and titratable acidity in the kimchi beverage base during co-fermentation. (<b>A</b>) Change in pH according to MSG content. (<b>B</b>) Change in titratable acidity according to MSG content. MSG content added: M3 (3% MSG); M5 (5% MSG); M7 (7% MSG). The results present the mean ± standard deviation (<span class="html-italic">n</span> = 3); Different letters (<sup>A–C</sup>) and (<sup>a–d</sup>) indicate statistically significant differences between the conditions and fermentation times, respectively, (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Thin-layer chromatography (TLC) of kimchi beverage base during co-fermentation. MSG content added: M3 (3% MSG); M5 (5% MSG); M7 (7% MSG).</p>
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14 pages, 2345 KiB  
Article
Study of Ultrasound-Assisted Low-Pressure Closed Acid Digestion Method for Trace Element Determination in Rock Samples by Inductively Coupled Plasma Mass Spectrometry
by Xijuan Tan, Yunxiu Ren, Ting Liang and Denghong Wang
Molecules 2025, 30(2), 342; https://doi.org/10.3390/molecules30020342 - 16 Jan 2025
Viewed by 435
Abstract
In this paper, a method of ultrasound-assisted low-pressure closed acid digestion followed by inductively coupled plasma mass spectrometry (ICP-MS) analysis was proposed for trace element quantification in rock samples. By using 1.5 mL of a binary acid mixture of HNO3–HF with [...] Read more.
In this paper, a method of ultrasound-assisted low-pressure closed acid digestion followed by inductively coupled plasma mass spectrometry (ICP-MS) analysis was proposed for trace element quantification in rock samples. By using 1.5 mL of a binary acid mixture of HNO3–HF with a ratio of 2:1, rock powder samples of 50 mg were completely decomposed in 12 h at 140 °C after 4 h of ultrasonic treatment with or without pressure relief procedure. The element extraction efficiency of this method was evaluated via the yielded relative errors (REs) of the trace elements in a series of geological standard reference materials (SRMs) with compositions from basic to acidic. It was found that the contents of trace elements (i.e., 36 metal elements from Li to U) in basalt BCR-2, diabase W-2a, andesite AGV-2, granodiorite GSP-2, and granite GSR-1 were comparable with the reported reference values, giving REs with absolute values less than 10%. It was also found that clear solutions without sample powder residues by naked-eye observation can be obtained when using the low-pressure closed decomposition method without ultrasonic pretreatment. The quantification results, however, were found to be negatively biased for most of the studied trace elements, and, in particular, the content bias of Zr in SRM GSP-2 was down to −86.28% due to the low extraction efficiency of refractory minerals of the low-pressure closed digestion method. By applying this proposed digestion strategy, the decomposition property of the ternary combination of HNO3–HF–mannitol in terms of trace element quantification accuracy was also investigated. Results showed that the concentrations of trace elements in the studied SRMs were consistent with the reference values, giving REs within ±6.94%, which revealed that there was no deterioration of extraction efficiencies of trace elements and neglected mass interferences from mannitol. This study demonstrated the essential role of ultrasound irradiation in rock sample decomposition to achieve the high extraction efficiency of trace elements under a low-pressure environment, and the developed approach with promising future applications in geoscience exhibited considerable merits, including a high extraction efficiency, feasible digestion process, less time consumption, and lower safety associated risks. Full article
(This article belongs to the Section Analytical Chemistry)
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<p>Ratios of quantified contents of trace elements to reference values in silicate SRMs by different digestion methods. Here, five silicate standard materials including BCR-2 (<b>a</b>), W-2a (<b>b</b>), AGV-2 (<b>c</b>), GSP-2 (<b>d</b>), and GSR-1 (<b>e</b>) were applied in this study. For each method, four parallel specimens of one silicate standard material with a sample weight of 50 mg (±0.5 mg) were digested. The trace element contents utilized to yield the ratios were the average of ICP-MS quantification results of the four specimens.</p>
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<p>Results of REs of trace element quantification by using the low-pressure closed acid digestion method. Here, five silicate standard materials including BCR-2, W-2, AGV-2, GSP-2, and GSR-1 of 50 mg (±0.5 mg) were decomposed by the low-pressure closed acid digestion method without ultrasound sonication treatment. The values of REs were obtained by the math calculation form of (C − C<sub>reference</sub>)/C<sub>reference</sub> × 100%.</p>
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14 pages, 3097 KiB  
Article
Role of Carrot (Daucus carota L.) Storage Roots in Drought Stress Adaptation: Hormonal Regulation and Metabolite Accumulation
by Kyoung Rok Geem, Ye-Jin Lee, Jeongmin Lee, Dain Hong, Ga-Eun Kim and Jwakyung Sung
Metabolites 2025, 15(1), 56; https://doi.org/10.3390/metabo15010056 - 16 Jan 2025
Viewed by 696
Abstract
Background: Drought stress has become one of the biggest concerns in threating the growth and yield of carrots (Daucus carota L.). Recent studies have shed light on the physiological and molecular metabolisms in response to drought in the carrot plant; however, tissue-specific [...] Read more.
Background: Drought stress has become one of the biggest concerns in threating the growth and yield of carrots (Daucus carota L.). Recent studies have shed light on the physiological and molecular metabolisms in response to drought in the carrot plant; however, tissue-specific responses and regulations are still not fully understood. Methods: To answer this curiosity, this study investigated the interplay among carrot tissues, such as leaves (L); storage roots (SRs); and lateral roots (LRs) under drought conditions. This study revealed that the SRs played a crucial role in an early perception by upregulating key genes, including DcNCED3 (ABA biosynthesis) and DcYUCCA6 (auxin biosynthesis). The abundance of osmolytes (proline; GABA) and carbohydrates (sucrose; glucose; fructose; mannitol; and inositol) was also significantly increased in each tissue. In particular, LRs accumulated high levels of these metabolites and promoted growth under drought conditions. Conclusions: Our findings suggest that the SR acts as a central regulator in the drought response of carrots by synthesizing ABA and auxin, which modulate the accumulation of metabolites and growth of LRs. This study provides new insights into the mechanisms of tissue-specific carrot responses to drought tolerance, emphasizing that the SR plays a key role in improving drought resistance. Full article
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<p><b>Growth and biomass production of carrot plant under different water potentials.</b> (<b>A</b>) Phenotype of 40-day-old carrots (<span class="html-italic">Daucus carota L.</span>) treated with 0 and −1.5 Mpa (10% of PEG-6000). Scale bar = 5 cm. Measurement of (<b>B</b>) shoot length, (<b>C</b>) root length, (<b>D</b>) shoot dry weight, and (<b>E</b>) lateral root dry weight. Statistical significance was determined by a T-test: * <span class="html-italic">p</span> &lt; 0.05. ns indicates non-significant difference.</p>
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<p><b>Proline contents under different water potentials.</b> The content of proline was measured in the leaves (L), storage root (SR), and lateral root (LR) from 40-day-old carrots treated with 0 and −1.5 Mpa (10% of PEG-6000). Measurement of proline contents in (<b>A</b>) leaves, (<b>B</b>) storage roots, and (<b>C</b>) lateral roots at 3 and 10 DAT. Statistical significance was determined by a T-test: * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. "ns" indicates no significant difference in proline levels in the leaf, storage roots, and lateral roots of carrots under different water potentials.</p>
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<p><b>5-Oxoproline and GABA contents in leaf, SR, and LR under different water potentials.</b> 5-Oxoproline and GABA were extracted from 40-day-old carrots treated with 0 and −1.5 Mpa (10% of PEG-6000) at 10 DAT. Measurement of (<b>A</b>) 5-Oxoproline contents, and (<b>B</b>) GABA contents in leaf, SR and LR at 10 DAT. Statistical significance was determined by T-test: *** <span class="html-italic">p</span> &lt; 0.001 in the leaf, storage roots, and lateral roots of carrots under different water potentials.</p>
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<p><b>Abundance of selected carbohydrates in leaf, SR, and LR under different water potentials.</b> All carbohydrates were extracted from 40-day-old carrots treated with 0 and −1.5 Mpa (10% of PEG-6000) at 10 DAT. Measurement of (<b>A</b>) Sucrose, (<b>B</b>) Glucose, (<b>C</b>) Fructose, (<b>D</b>) Mannitol, and (<b>E</b>) Myo-inositol contents in leaf, SR and LR at 10 DAT. Statistical significance was determined by T-test: ** <span class="html-italic">p</span> &lt; 0.01; and *** <span class="html-italic">p</span> &lt; 0.001 in the leaf, storage roots, and lateral roots of carrots under different water potentials. "ns" indicates no significant difference.</p>
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<p><b>Relative expression of ABA-related genes under different water potentials.</b> Total RNA was extracted from leaves(L), storage root (SR), and lateral root (LR) from 40-day-old carrots treated with 0 and −1.5 Mpa (10% of PEG-6000), and analyzed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The expression levels of <span class="html-italic">DcNCED3</span>, <span class="html-italic">DcPYL9</span>, and <span class="html-italic">DcSnRK2</span> were measured at 3 DAT in (<b>A</b>) L, (<b>B</b>) LR, and (<b>C</b>) SR and at 10 DAT in (<b>D</b>) L, (<b>E</b>) LR, and (<b>F</b>) SR. Statistical significance was determined by T-test: * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; and *** <span class="html-italic">p</span> &lt; 0.001 in the leaf, storage roots, and lateral roots of carrots under different water potentials.</p>
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<p><b>Relative expression of Auxin-related genes under different water potentials.</b> Total RNA was extracted from leaves(L), storage root (SR), and lateral root (LR) from 40-day-old carrots treated with 0 and −1.5 Mpa (10% of PEG-6000), and analyzed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The expression levels of <span class="html-italic">DcLAX1</span>, <span class="html-italic">DcYUCCA6</span>, and <span class="html-italic">DcARF8</span> were measured at 3 DAT in (<b>A</b>) L, (<b>B</b>) LR, and (<b>C</b>) SR and at 10 DAT in (<b>D</b>) L, (<b>E</b>) LR, and (<b>F</b>) SR. Statistical significance was determined by T-test: * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; and *** <span class="html-italic">p</span> &lt; 0.001 in the leaf, storage roots, and lateral roots of carrots under different water potentials.</p>
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18 pages, 12492 KiB  
Article
Untargeted Metabolomics Reveals Key Differences Between Yak, Buffalo, and Cow Colostrum Based on UHPLC-ESI-MS/MS
by Yuzhuo Wang, Changhui Li, Jiaxiang Huang, Qingkun Zeng, Ling Li, Pan Yang, Pengjie Wang, Min Chu, Jie Luo, Fazheng Ren and Hao Zhang
Foods 2025, 14(2), 232; https://doi.org/10.3390/foods14020232 - 13 Jan 2025
Viewed by 727
Abstract
Background: Colostrum, abundant in immunoglobulins and growth factors, plays a vital role in supporting immunity. Both yak and buffalo milk are characterized by their high protein and fat content. However, the metabolomic profiles of yak colostrum (YC), buffalo colostrum (BC), and bovine colostrum [...] Read more.
Background: Colostrum, abundant in immunoglobulins and growth factors, plays a vital role in supporting immunity. Both yak and buffalo milk are characterized by their high protein and fat content. However, the metabolomic profiles of yak colostrum (YC), buffalo colostrum (BC), and bovine colostrum (CC) remain largely unexplored. The objective of this study is to identify unique metabolites that may impact the nutritional value of colostrum. Methods: This study employed ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) for untargeted metabolomics analysis of YC, BC, and CC. Results: The analysis revealed 97, 70, and 75 differentially expressed metabolites in the YC-CC, BC-CC, and YC-BC comparisons, respectively. In comparison to CC, both YC and BC shared common features, including reduced choline levels and elevated O-acetylcarnitine. Moreover, metabolites such as 2-hydroxy-6-pentadecylbenzoic acid, DL-glycerol-1-phosphate, thiamine, L-carnitine, methyl β-D-galactoside, and uridine diphosphate (UDP) were identified as potential biomarkers for YC, while 21-deoxycortisol, D-synephrine, uridine, mannitol-1-phosphate, nonadecanoic acid, and perillic acid were specific to BC. Conclusions: YC has greater advantages in energy supply, antioxidant activity, immune regulation, and cell homeostasis, and BC holds unique significance in physical development and energy balance regulation. These findings provide valuable insights, enabling the selection of unique bioactive metabolites to develop targeted functional foods from colostrum, catering to diverse nutritional needs. Full article
(This article belongs to the Topic Advances in Animal-Derived Non-Cow Milk and Milk Products)
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<p>Multivariate analysis of yak colostrum (YC), buffalo colostrum (BC), and cow colostrum (CC). (<b>A</b>,<b>B</b>) Principal component analysis (PCA) score plots in positive and negative ion modes, respectively. (<b>C</b>,<b>D</b>) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plots in positive and negative ion modes, respectively. (<b>E</b>,<b>F</b>) OPLS-DA permutation test plots in positive and negative ion modes, respectively. (<b>G</b>,<b>H</b>) Correlation heatmaps of samples in positive and negative ion modes, respectively. *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Pairwise analysis of yak colostrum (YC), buffalo colostrum (BC), and cow colostrum (CC). (<b>A</b>) Volcano plot of differentially expressed metabolites (DEMs) between YC and CC. (<b>B</b>) Subclass composition of DEMs between YC and CC. (<b>C</b>) KEGG enrichment analysis presented as a mulberry map and bubble map for DEMs between YC and CC. (<b>D</b>) Volcano plot of DEMs between BC and CC. (<b>E</b>) Subclass composition of DEMs between BC and CC. (<b>F</b>) KEGG enrichment analysis as a mulberry map and bubble map for DEMs between BC and CC. (<b>G</b>) Volcano plot of DEMs between YC and BC. (<b>H</b>) Subclass composition of DEMs between YC and BC. (<b>I</b>) KEGG enrichment analysis in mulberry and bubble maps for DEMs between YC and BC.</p>
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<p>Clustering and distribution of all differentially expressed metabolites (DEMs) from pairwise analysis of yak colostrum (YC), buffalo colostrum (BC), and cow colostrum (CC). (<b>A</b>) Heatmap showing all DEMs across YC, BC, and CC. (<b>B</b>) Upset plot displaying the overlap and unique DEMs among YC, BC, and CC.</p>
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<p>Metabolite markers of yak colostrum (YC). (<b>A</b>) Z-score map of differentially expressed metabolites (DEMs) with higher expression in YC compared to BC and CC. (<b>B</b>) Relative content of 2-hydroxy-6-pentadecylbenzoic acid, DL-glycerol-1-phosphate, thiamine, L-carnitine, methyl β-D-galactoside, and uridine diphosphate (UDP) across YC, BC, and CC. (<b>C</b>) Receiver operating characteristic (ROC) curves for 2-hydroxy-6-pentadecylbenzoic acid, DL-glycerol-1-phosphate, thiamine, L-carnitine, methyl β-D-galactoside, and UDP. (<b>D</b>) KEGG pathway enrichment analysis of DEMs with differential expression in YC compared to BC and CC.</p>
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<p>Metabolite markers of buffalo colostrum (BC). (<b>A</b>) Z-score map of differentially expressed metabolites (DEMs) with higher expression in BC compared to YC and CC. (<b>B</b>) Relative content of 21-deoxycortisol, D-synephrine, uridine, mannitol-1-phosphate, nonadecanoic acid, and perillic acid across YC, BC, and CC. (<b>C</b>) The receiver operating characteristic (ROC) curves for 21-deoxycortisol, D-synephrine, uridine, mannitol-1-phosphate, nonadecanoic acid, and perillic acid. (<b>D</b>) KEGG pathway enrichment analysis of DEMs with differential expression in BC compared to YC and CC.</p>
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17 pages, 5618 KiB  
Article
Comprehensive Identification of AREB Gene Family in Populus euphratica Oliv. and Functional Analysis of PeAREB04 in Drought Tolerance
by Binglei Liu, Jianhao Sun, Chen Qiu, Xiaoli Han and Zhijun Li
Int. J. Mol. Sci. 2025, 26(2), 518; https://doi.org/10.3390/ijms26020518 - 9 Jan 2025
Viewed by 504
Abstract
The transcription factors in the ABA Response Element Binding (AREB) protein family were differentially regulated under multiple stress conditions; however, functional analyses of AREB in Populus euphratica Oliv. had not been conducted previously. In the present study, the comprehensive identification of the P. [...] Read more.
The transcription factors in the ABA Response Element Binding (AREB) protein family were differentially regulated under multiple stress conditions; however, functional analyses of AREB in Populus euphratica Oliv. had not been conducted previously. In the present study, the comprehensive identification of the P. euphratica AREB gene family and the function of PeAREB04 in response to drought stress in P. euphratica were elucidated. A comprehensive analysis of the PeAREB family was first performed, followed by the determination of their expression patterns under drought stress. Bioinformatics analysis revealed that thirteen AREB genes were identified across the P. euphratica genome, with these genes distributed across eight chromosomes in a seemingly random pattern. Phylogenetic analysis indicated that the PeAREB genes could be categorized into four distinct branches. Cis-acting element analysis revealed that most PeAREB genes contained multiple hormone- and stress-responsive elements. Transcriptomic sequencing of P. euphratica seedlings under drought stress showed that most PeAREB genes responded rapidly to drought stress in either the leaves or roots. One gene, PeAREB04, was selected for further functional validation due to its significant upregulation in both leaves and roots under drought stress. Overexpression of PeAREB04 in Arabidopsis thaliana resulted in a high survival rate, reduced water loss in isolated leaves, and a significant reduction in stomatal aperture under natural drought conditions. Drought stress simulations using mannitol further demonstrated that overexpression of PeAREB04 significantly enhanced root elongation. These findings indicate that the identification of the PeAREB gene family and the characterization of PeAREB04’s role in drought stress have been largely accomplished. Furthermore, the PeAREB04 gene demonstrates considerable potential as a key target for future genetic engineering strategies aimed at enhancing plant drought resistance. Full article
(This article belongs to the Special Issue Transcriptional Regulation in Plant Development: 2nd Edition)
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<p>Chromosomal localization and gene family replication type of <span class="html-italic">PeAREB</span> genes. (<b>A</b>) Analysis of the chromosomal localisation of <span class="html-italic">PeAREBs</span>. (<b>B</b>) Analysis of <span class="html-italic">PeAREB</span> gene family replication types.</p>
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<p>Phylogenetic relationship, gene conserved domain, gene structure and protein conserved domain of PeAREBs conserved protein motifs. (<b>A</b>) Neighbor-joining(NJ) phylogenetic tree of 13 PeAREB proteins; conserved domain of <span class="html-italic">PeAREB</span> gene family; exon/intron structure of <span class="html-italic">PeAREBs</span>. (<b>B</b>) Predicting the conserved domain of PeAREBs protein. Different colour boxes represent conserved domains, gene structures and motifs of different gene families.</p>
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<p>The phylogeny and collinearity of multi-species <span class="html-italic">AREB</span> genes, and intraspecific collinearity of <span class="html-italic">PeAREBs</span>. (<b>A</b>) <span class="html-italic">P. euphratica</span>, <span class="html-italic">P. pruinosa</span>, <span class="html-italic">S. purpurea</span> and <span class="html-italic">A. thaliana</span> species collinearity. Numbers represent chromosome numbers. The cyan short bars indicate <span class="html-italic">P. euphratica</span> chromosomes, while the yellow short bars at different positions represent <span class="html-italic">A. thaliana</span>, <span class="html-italic">P. pruinosa</span>, and <span class="html-italic">S. purpurea</span>. (<b>B</b>) Intraspecific collinearity of <span class="html-italic">P. euphratica</span>.</p>
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<p>Phylogenetic analysis of AREBs in <span class="html-italic">P. euphratica</span>, <span class="html-italic">P. pruinosa</span>, <span class="html-italic">S. purpurea</span> and <span class="html-italic">A. thaliana</span>.</p>
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<p>Predicted cis-acting elements in the promoter regions of <span class="html-italic">PeAREB</span> gene family members.</p>
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<p>Expression patterns of <span class="html-italic">PeAREB</span> genes.</p>
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<p>Nuclear localization of the 35S::<span class="html-italic">PeAREB04</span>-GFP protein in tobacco leaf epidermal cells. The fluorescence images of PeAREB04 (35S::<span class="html-italic">PeAREB04</span>-GFP), nuclear localization signal (NLS-mCherry) and merged image (35S::<span class="html-italic">PeAREB04</span>-GFP/NLS-mCherry) were sequentially displayed from left to right. (Scale bar = 20 μm).</p>
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<p>Drought tolerance assays for <span class="html-italic">PeAREB04</span> overexpression lines (T3). (<b>A</b>) Phenotypes observed in the drought tolerance assays. (<b>B</b>) Survival rates of WT and OE lines. (<b>C</b>) Water loss rates of detached leaves from WT and OE lines. (<b>D</b>) Microscope images showing stomatal openings in WT and three overexpression lines (OE-1, OE-2 and OE-3) under control and drought stress conditions. Scale bar = 5 μm. (<b>E</b>) Stomatal opening measurements in leaves of WT, OE-1, OE-2 and OE-3 under control and drought stress conditions. Each treatment was randomly photographed to select 30 stomata. (<b>F</b>) Phenotype of WT and OE lines under mannitol treatment. Scale bar = 1 cm. (<b>G</b>) Root length measurements of <span class="html-italic">Arabidopsis</span> seedlings under control and mannitol treatment conditions. Different letters indicate significant differences based on Duncan’s least significant range test (<span class="html-italic">p</span> &lt; 0.05).</p>
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