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27 pages, 1920 KiB  
Review
Recent Technological Advancements for Identifying and Exploiting Novel Sources of Pest and Disease Resistance for Peanut Improvement
by Akshaya Kumar Biswal, Peggy Ozias-Akins and Carl Corley Holbrook
Agronomy 2024, 14(12), 3071; https://doi.org/10.3390/agronomy14123071 (registering DOI) - 23 Dec 2024
Abstract
Peanut, also known as groundnut (Arachis hypogaea L.), is an important oilseed and food crop globally, contributing significantly to the economy and food security. However, its productivity is often hampered by pests and diseases. Traditional breeding methods have been used to develop [...] Read more.
Peanut, also known as groundnut (Arachis hypogaea L.), is an important oilseed and food crop globally, contributing significantly to the economy and food security. However, its productivity is often hampered by pests and diseases. Traditional breeding methods have been used to develop resistant cultivars, but these are often time-consuming and labor-intensive. Recent technological advancements have revolutionized the identification of novel resistance sources and the development of resistant peanut cultivars. This review explores the latest techniques and approaches used in peanut breeding for pest and disease resistance, focusing on the identification of resistance loci and their incorporation into peanut using marker-assisted selection (MAS) and genomic tools. Next-generation sequencing (NGS) technologies, bioinformatics pipelines, comparative genomics, and transcriptomics have helped identify a plethora of candidate genes involved in pest resistance. However, peanut lags behind other cereal crops in terms of phenomics and precision genetic techniques for their functional validation. In conclusion, recent technological advancements have significantly improved the efficiency and precision of peanut breeding for pest and disease resistance and hold great promise for developing durable and sustainable resistance in peanut cultivars, ultimately benefiting peanut farmers and consumers globally. Full article
(This article belongs to the Special Issue Pest Control Technologies Applied in Peanut Production Systems)
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<p>Application of recent technologies in peanut breeding. This figure illustrates the synergy between traditional breeding techniques and modern genomic tools, underscoring the potential for developing pest and disease-resistant peanut cultivars with enhanced adaptability: (1) germplasm collection includes wild-type peanut plants, landraces, and breeding populations, serving as the genetic foundation for breeding efforts, (2) schematic representation of a breeding population, (3) identification and selection of lines exhibiting resistance to specific diseases, (4) accelerated generation advancement through controlled environment techniques, facilitating faster selection cycles, (5) establishment of recombinant inbred lines (RILs), near-isogenic lines (NILs), multi-parent advanced generation inter-cross (MAGIC) populations, and nested association mapping (NAM) populations to enhance genetic diversity and study complex traits, (6) use of PCR, SNP arrays, and whole-genome sequencing to characterize genetic variation, (7) analysis of marker–trait or SNP–trait associations, QTL mapping and development of molecular markers for key agronomic traits, (8) evaluation of breeding lines across different environments to assess genotype × environment interactions through multilocation trials, (9) computational approaches to predict candidate genes associated with desirable traits, (10) application of molecular markers to select and develop disease-resistant cultivars, (11) use of CRISPR-mediated genome editing and other reverse genetic tools for functional validation of candidate genes and development of improved germplasm, and (12) integration of genomic data to predict and select superior parents for breeding programs, and (13) an improved peanut variety developed through MAS, genome editing, or other reverse genetics technologies.</p>
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16 pages, 8703 KiB  
Article
Disrupted Lipid Metabolism, Cytokine Signaling, and Dormancy: Hallmarks of Doxorubicin-Resistant Triple-Negative Breast Cancer Models
by Radhakrishnan Vishnubalaji and Nehad M. Alajez
Cancers 2024, 16(24), 4273; https://doi.org/10.3390/cancers16244273 (registering DOI) - 23 Dec 2024
Abstract
Background: Chemoresistance in triple-negative breast cancer (TNBC) presents a significant clinical hurdle, limiting the efficacy of treatments like doxorubicin. This study aimed to explore the molecular changes associated with doxorubicin resistance and identify potential therapeutic targets to overcome this resistance, thereby improving treatment [...] Read more.
Background: Chemoresistance in triple-negative breast cancer (TNBC) presents a significant clinical hurdle, limiting the efficacy of treatments like doxorubicin. This study aimed to explore the molecular changes associated with doxorubicin resistance and identify potential therapeutic targets to overcome this resistance, thereby improving treatment outcomes for TNBC patients. Methods: Doxorubicin-resistant (DoxR) TNBC models (MDA-MB-231 and BT-549) were generated by exposing cells to increasing concentrations of doxorubicin. RNA sequencing (RNA-Seq) was performed using the Illumina platform, followed by bioinformatics analysis with CLC Genomics Workbench and iDEP. Functional assays assessed proliferation, sphere formation, migration, and cell cycle changes. Protein expression and phosphorylation were confirmed via Western blotting. Pathway and network analyses were conducted using Ingenuity Pathway Analysis (IPA) and STRING, while survival analysis was performed using Kaplan–Meier Plotter database. Results: DoxR cells exhibited reduced proliferation, sphere formation, and migration, but showed enhanced tolerance to doxorubicin. Increased CHK2 and p53 phosphorylation indicated cellular dormancy as a resistance mechanism. RNA-Seq analysis revealed upregulation of cytokine signaling and stress-response pathways, while cholesterol and lipid biosynthesis were suppressed. Activation of the IL1β cytokine network was prominent in DoxR cells, and CRISPR-Cas9 screens data identified dependencies on genes involved in rRNA biogenesis and metabolism. A 27-gene signature associated with doxorubicin resistance was linked to worse clinical outcomes in a large breast cancer cohort (HR = 1.76, FDR p < 2.0 × 10−13). Conclusions: This study uncovers potential therapeutic strategies for overcoming TNBC resistance, including dormancy reversal and targeting onco-ribosomal pathways and cytokine signaling networks, to improve the efficacy of doxorubicin-based treatments. Full article
(This article belongs to the Special Issue Molecular Insights into Drug Resistance in Cancer)
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Graphical abstract

Graphical abstract
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<p>Proliferation and morphological changes in parental and DoxR TNBC cells following doxorubicin treatment. (<b>A</b>) Clonogenic survival assay (CFU) showing dose-dependent suppression of proliferation in parental TNBC cells (MDA-MB-231 and BT-549) treated with 12.5 nM and 25 nM doxorubicin, while DoxR cells maintained higher proliferation rates. (<b>B</b>) Quantitative analysis of CFU potential reveals reduced inhibition of cell proliferation in DoxR MDA-MB-231 and BT-549 cells compared to parental cells at both doxorubicin concentrations. Data are presented as mean  ±  S.E.M., n  =  6. n.s., not significant; * <span class="html-italic">p</span>  &lt;  0.05; ** <span class="html-italic">p</span>  &lt;  0.005; **** <span class="html-italic">p</span>  &lt;  0.00005. Two-way ANOVA testing was employed. (<b>C</b>) Acridine orange/ethidium bromide (AO/EtBr) staining of parental and DoxR cells, demonstrating the presence of necrotic cells (red) and morphological abnormalities in response to doxorubicin in parental cells.</p>
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<p>Suppressed migration and organotypic growth in DoxR TNBC models. Representative organotypic images for MDAMB-231 (<b>A</b>) or BT-549 (<b>B</b>) DoxR and control TNBC cells. (<b>C</b>) Spheroid formation assay showing reduced spheroid formation capacity in DoxR TNBC cells compared to parental cells. Migration assay illustrates a significant reduction in the migration of DoxR MDA-MB-231 (<b>D</b>) and BT-549 (<b>E</b>) cells compared to parental cells. Quantification of relative wound areas (%) are shown below. Data are presented as mean ± S.D., n = 2. n.s., not significant; ** <span class="html-italic">p</span>  &lt;  0.005; *** <span class="html-italic">p</span>  &lt;  0.0005.</p>
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<p>Alterations in cell cycle regulation in DoxR TNBC cells. (<b>A</b>) Flow cytometry analysis of cell cycle distribution in parental and DoxR cells post-doxorubicin treatment on day 3. (<b>B</b>) Quantification of cell cycle distribution form (<b>A</b>). Data are presented as mean ± S.D., n = 2. (<b>C</b>) Annexin V staining of control and DoxR TNBC cells at the indicated doxorubicin concentrations on day 3. (<b>D</b>) Representative Western blot analysis showing phosphorylated Retinoblastoma protein (p-Rb), phosphorylated ChK kinases, and p53 in DoxR TNBC cells. Quantifications of band intensity are shown on the right panel. Data are presented as mean ± S.D., n = 2. n.s., not significant; * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.005.</p>
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<p>Molecular profiling of DoxR TNBC models. (<b>A</b>) Heatmap of differentially expressed genes (DEGs) showing 231 upregulated and 420 downregulated genes in DoxR cells compared to parental TNBC models. (<b>B</b>) Hierarchical clustering of enriched gene ontology (GO) biological processes, with upregulated genes associated with defense and immune responses and downregulated genes involved in cholesterol synthesis. (<b>C</b>) RT-qPCR validation of selected upregulated and downregulated genes in DoxR MDA-MB-231 cells. Data are presented as mean  ±  S.E.M., n = 9. * <span class="html-italic">p</span>  &lt;  0.05, *** <span class="html-italic">p</span>  &lt;  0.0005. (<b>D</b>) Disease and function heatmap depicting activated (orange) and suppressed (blue) categories in DoxR vs. control TNBC cells. (<b>E</b>) Upstream regulator analysis illustrating IL1β as a key driver of gene expression changes in DoxR TNBC models. n.s., not significant; ** <span class="html-italic">p</span> &lt; 0.005; **** <span class="html-italic">p</span> &lt; 0.00005.</p>
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<p>Dependency map of DoxR TNBC models highlighting the role of ribosomal RNA. (<b>A</b>) CRISPR-Cas9 functional screen data from DepMap identifying essentiality of 27 genes among 211 upregulated genes in DoxR TNBC models. (<b>B</b>) The effects of each identified gene in the panel of 22 TNBC models are depicted as a heatmap. The heatmap represents the dependency scores calculated by DepMap for the identified genes. (<b>C</b>) Kaplan–Meier plot showing that the identified 27-gene signature is associated with poor relapse-free survival (RFS) in breast cancer patients. (<b>D</b>) GeneMANIA analysis showing significant enrichment of the identified genes in functional networks associated with rRNA biogenesis and metabolism, underscoring their critical role in TNBC pathophysiology. (<b>E</b>) Functional enrichment among the DoxR-derived signature employment GeneMANIA.</p>
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19 pages, 337 KiB  
Article
Auditors’ Perceptions of the Triggers and Obstacles of Continuous Auditing and Its Impact on Auditor Independence: Insights from Egypt
by Laila Mohamed Alshawadfy Aladwey and Samar El Sayad
J. Risk Financial Manag. 2024, 17(12), 578; https://doi.org/10.3390/jrfm17120578 (registering DOI) - 23 Dec 2024
Abstract
Our study explores auditors’ perceptions of the triggers and hurdles of implementing continuous auditing (CA) in Egypt. It also explores auditors’ perceptions of the impact of CA on their independence. A survey of ninety-five auditors working in Big Four and non-Big Four firms [...] Read more.
Our study explores auditors’ perceptions of the triggers and hurdles of implementing continuous auditing (CA) in Egypt. It also explores auditors’ perceptions of the impact of CA on their independence. A survey of ninety-five auditors working in Big Four and non-Big Four firms was conducted to gather data. Descriptive statistics and the Friedman test were used to test our hypotheses. In addition, using the Mann–Whitney U test, we delve deeper into auditors’ perceptions to examine differences across audit firm types. The results reveal that addressing the increasing demand of stakeholders for real-time reporting and enhancing the quality of financial reporting significantly affect auditors’ perceptions of the triggers for adopting CA. In addition, the lack of standards related to CA and the high cost of implementation significantly affect auditors’ perceptions of the obstacles to implementing CA. The lack of clear guidelines regarding the work required in CA and auditing data that the auditors have previously corrected during the CA process is perceived by auditors as among the most significant factors that can impair their independence. The significance of this study stems from the fact that it is one of the few studies to explore continuous auditing practices in developing countries. To the best of our knowledge, this study is one of the first to investigate how CA affects auditor independence in developing countries. Full article
(This article belongs to the Special Issue Auditing, Corporate Governance and Financial Reporting Quality)
17 pages, 4090 KiB  
Article
Crack Sealing in Concrete with Biogrout: Sustainable Approach to Enhancing Mechanical Strength and Water Resistance
by Jian Wang, Shengjie Ji, Shuguang Huang, Zihang Jiang, Siqi Wang, Huaiqi Zhang, Zijian Wang and Junfei Zhang
Materials 2024, 17(24), 6283; https://doi.org/10.3390/ma17246283 (registering DOI) - 23 Dec 2024
Abstract
Concrete, as the most widely used construction material globally, is prone to cracking under the influence of external factors such as mechanical loads, temperature fluctuations, chemical corrosion, and freeze–thaw cycles. Traditional concrete crack repair methods, such as epoxy resins and polymer mortars, often [...] Read more.
Concrete, as the most widely used construction material globally, is prone to cracking under the influence of external factors such as mechanical loads, temperature fluctuations, chemical corrosion, and freeze–thaw cycles. Traditional concrete crack repair methods, such as epoxy resins and polymer mortars, often suffer from a limited permeability, poor compatibility with substrates, and insufficient long-term durability. Microbial biogrouting technology, leveraging microbial-induced calcium carbonate precipitation (MICP), has emerged as a promising alternative for crack sealing. This study aimed to explore the potential of Bacillus pasteurii for repairing concrete cracks to enhance compressive strength and permeability performance post-repair. Experiments were conducted to evaluate the bacterial growth cycle and urease activity under varying concentrations of Ca2+. The results indicated that the optimal time for crack repair occurred 24–36 h after bacterial cultivation. Additionally, the study revealed an inhibitory effect of high calcium ion concentrations on urease activity, with the optimal concentration identified as 1 mol/L. Compressive strength and water absorption tests were performed on repaired concrete specimens. The compressive strength of specimens with cracks of varying dimensions improved by 4.01–11.4% post-repair, with the highest improvement observed for specimens with 1 mm wide and 10 mm deep cracks, reaching an increase of 11.4%. In the water absorption tests conducted over 24 h, the average mass water absorption rate decreased by 31.36% for specimens with 0.5 mm cracks, 29.06% for 1 mm cracks, 27.9% for 2 mm cracks, and 28.2% for 3 mm cracks. X-ray diffraction (XRD) and scanning electron microscopy (SEM) analyses confirmed the formation of dense calcium carbonate precipitates, with the SEM–EDS results identifying calcite and vaterite as the predominant self-healing products. This study underscores the potential of MICP-based microbial biogrouting as a sustainable and effective solution for enhancing the mechanical and durability properties of repaired concrete. Full article
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<p>Schematic diagram of MICP repairing concrete cracks.</p>
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<p>The experimental process.</p>
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<p>The process of the water absorption experiment.</p>
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<p>Growth conditions of <span class="html-italic">Bacillus pasteurii</span> and urease activity assay: (<b>a</b>) the growth of <span class="html-italic">Bacillus pasteurii</span> within 60 h and (<b>b</b>) the variation in urease activity with changes in Ca<sup>2+</sup>.</p>
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<p>Visualization and UCS variations in repaired concrete cracks: (<b>a</b>) comparison of mineralization repair for cracks of different widths before and after treatment, (<b>b</b>) compressive strength before and after crack repair, and (<b>c</b>) increase ratio in compressive strength after repair.</p>
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<p>Water absorption and rate of change in water absorption of different crack specimens over time: (<b>a</b>–<b>d</b>) the variation in water absorption over time and crack depth for crack widths of 0.5 mm, 1 mm, 2 mm, and 3 mm before and after repair, (<b>e</b>,<b>f</b>) the water absorption rates of cracks with a width of 2 mm at different depths after 24 h and 48 h, and (<b>g</b>,<b>h</b>) the water absorption rates of cracks with a width of 2 mm at different depths after 24 h and 48 h.</p>
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<p>EDS, SEM, and XRD results of mineralization products: (<b>a</b>) EDS mapping of mineralization products, (<b>b</b>) microstructure and morphology of mineralization products and junction, and EDS line scans of junction, and (<b>c</b>) mineralization products and their XRD patterns.</p>
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15 pages, 10134 KiB  
Article
Investigation of Calcium Phosphate-Based Biopolymer Composite Scaffolds for Bone Tissue Engineering
by Monika Furko, Zsolt E. Horváth, Istvan Tolnai, Katalin Balázsi and Csaba Balázsi
Int. J. Mol. Sci. 2024, 25(24), 13716; https://doi.org/10.3390/ijms252413716 (registering DOI) - 22 Dec 2024
Abstract
We present a novel method for preparing bioactive and biomineralized calcium phosphate (mCP)-loaded biopolymer composite scaffolds with a porous structure. Two types of polymers were investigated as matrices: one natural, cellulose acetate (CA), and one synthetic, polycaprolactone (PCL). Biomineralized calcium phosphate particles were [...] Read more.
We present a novel method for preparing bioactive and biomineralized calcium phosphate (mCP)-loaded biopolymer composite scaffolds with a porous structure. Two types of polymers were investigated as matrices: one natural, cellulose acetate (CA), and one synthetic, polycaprolactone (PCL). Biomineralized calcium phosphate particles were synthesized via wet chemical precipitation, followed by the addition of organic biominerals, such as magnesium gluconate and zinc gluconate, to enhance the bioactivity of the pure CP phase. We compared the morphological and chemical characteristics of the two types of composites and assessed the effect of biomineralization on the particle structure of pure CP. The precipitated CP primarily consisted of nanocrystalline apatite, and the addition of organic trace elements significantly influenced the morphology by reducing particle size. FE-SEM elemental mapping confirmed the successful incorporation of mCP particles into both CA and PCL polymer matrices. Short-term immersion tests revealed that the decomposition rate of both composites is slow, with moderate and gradual ionic dissolution observed via ICP-OES measurements. The weight loss of the PCL-based composite during immersion was minimal, decreasing by only 0.5%, while the CA-based composite initially exhibited a slight weight increase before gradually decreasing over time. Full article
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Figure 1
<p>FE-SEM images of amorphous apatite (CP) (<b>a</b>) biomineralized (Mg, Zn added apatite (mCP) (<b>b</b>), pure cellulose acetate (<b>c</b>), pure PCL polymer (<b>d</b>), as well as their composites CA-mCP (<b>e</b>) and PCL-mCP (<b>f</b>). The parameters used in the preparation were kept consistent to ensure their comparability.</p>
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<p>FE-SEM images of amorphous apatite (CP) (<b>a</b>) biomineralized (Mg, Zn added apatite (mCP) (<b>b</b>), pure cellulose acetate (<b>c</b>), pure PCL polymer (<b>d</b>), as well as their composites CA-mCP (<b>e</b>) and PCL-mCP (<b>f</b>). The parameters used in the preparation were kept consistent to ensure their comparability.</p>
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<p>Scanning electron microscope image and the corresponding elemental mapping of biomineralized (Mg, Zn) calcium apatite.</p>
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<p>Scanning electron microscope image and the corresponding elemental mapping of PCL-mCP composite (<b>a</b>) and CA-mCP composite (<b>b</b>).</p>
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<p>XRD patterns of CP and mCP powders (<b>a</b>) prepared by wet chemical method and the PCL-mCP, CA.mCP composites (<b>b</b>).</p>
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<p>FE-SEM images on CA-mCP (<b>a</b>) and PCL-mCP (<b>c</b>) composites as prepared as well as CA-mCP (<b>b</b>) and PCL-mCP (<b>d</b>) composites after two weeks of immersion in saline solution.</p>
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<p>Sample weight changes during the two-week immersion period in saline solution at room temperature. Values are graphed as the mean ± standard deviation (<span class="html-italic">n</span> = 3). * indicates <span class="html-italic">p</span>  &lt;  0.05; ** indicates <span class="html-italic">p</span> &lt;  0.01.</p>
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<p>Cumulative concentrations of the dissolved bioactive ions from CA-mCP (<b>a</b>) and PCL-mCP (<b>b</b>) composites soaked in saline solution at room temperature. The values are normalized to the unit area of samples. All data points are presented as the mean ± standard deviation (<span class="html-italic">n</span> = 3).</p>
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16 pages, 3616 KiB  
Article
Altered Protein Kinase A-Dependent Phosphorylation of Cav1.2 in Left Ventricular Myocardium from Cacna1c Haploinsufficient Rat Hearts
by David Königstein, Hauke Fender, Jelena Plačkić, Theresa M. Kisko, Markus Wöhr and Jens Kockskämper
Int. J. Mol. Sci. 2024, 25(24), 13713; https://doi.org/10.3390/ijms252413713 (registering DOI) - 22 Dec 2024
Abstract
CACNA1C encodes the α1c subunit of the L-type Ca2+ channel, Cav1.2. Ventricular myocytes from haploinsufficient Cacna1c (Cacna1c+/−) rats exhibited reduced expression of Cav1.2 but an apparently normal sarcolemmal Ca2+ influx with an impaired response to sympathetic stress. We [...] Read more.
CACNA1C encodes the α1c subunit of the L-type Ca2+ channel, Cav1.2. Ventricular myocytes from haploinsufficient Cacna1c (Cacna1c+/−) rats exhibited reduced expression of Cav1.2 but an apparently normal sarcolemmal Ca2+ influx with an impaired response to sympathetic stress. We tested the hypothesis that the altered phosphorylation of Cav1.2 might underlie the sarcolemmal Ca2+ influx phenotype in Cacna1c+/− myocytes using immunoblotting of the left ventricular (LV) tissue from Cacna1c+/− versus wildtype (WT) hearts. Activation of cAMP-dependent protein kinase A (PKA) increases L-type Ca2+ current and phosphorylates Cav1.2 at serine-1928. Using an antibody directed against this phosphorylation site, we observed elevated phosphorylation of Cav1.2 at serine-1928 in LV myocardium from Cacna1c+/− rats under basal conditions (+110% versus WT). Sympathetic stress was simulated by isoprenaline (100 nM) in Langendorff-perfused hearts. Isoprenaline increased the phosphorylation of serine-1928 in Cacna1c+/− LV myocardium by ≈410%, but the increase was significantly smaller than in WT myocardium (≈650%). In conclusion, our study reveals altered PKA-dependent phosphorylation of Cav1.2 with elevated phosphorylation of serine-1928 under basal conditions and a diminished phosphorylation reserve during β-adrenergic stimulation. These alterations in the phosphorylation of Cav1.2 may explain the apparently normal sarcolemmal Ca2+ influx in Cacna1c+/− myocytes under basal conditions as well as the impaired response to sympathetic stimulation. Full article
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<p>Expression of Cav1.3 and Cavβ2 in LV myocardium from WT and <span class="html-italic">Cacna1c<sup>+/−</sup></span> rats. (<b>A</b>) Expression of Cav1.3 in LV myocardium from WT and <span class="html-italic">Cacna1c<sup>+/−</sup></span> rats. Values were normalized to the mean from WT. (<b>B</b>) Original Western blot images of 8 WT (+/+) and 8 <span class="html-italic">Cacna1c<sup>+/−</sup></span> (+/−) samples derived from two membranes. The protein used for normalization (GAPDH) derived from the same membrane is shown below the protein of interest. (<b>C</b>) Expression of Cavβ2 in LV myocardium from WT and <span class="html-italic">Cacna1c<sup>+/−</sup></span> rats. Values were normalized to the mean from WT. (<b>D</b>) Original Western blot images of 8 WT (+/+) and 8 <span class="html-italic">Cacna1c<sup>+/−</sup></span> (+/−) samples derived from two membranes. The protein used for normalization (GAPDH) derived from the same membrane is shown below the protein of interest. Circles represent number of animals: N = 8 (WT); N = 8 (<span class="html-italic">Cacna1c<sup>+/−</sup></span>); Student’s <span class="html-italic">t</span>-test, * <span class="html-italic">p</span> &lt; 0.05; ns = not significant. All original Western blot images from this series are shown in <a href="#app1-ijms-25-13713" class="html-app">Supplementary Materials</a>.</p>
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<p>Phosphorylation of Cav1.2 at S1928 following treatment of rat hearts with a phosphorylation (P) or dephosphorylation (De) solution. Original Western blot images showing results of LV homogenates from 8 Sprague-Dawley rat hearts treated with either a phosphorylation (P) or dephosphorylation (De) solution. <b>Top</b>: results obtained with the antibody directed against phosphorylated S1928 of Cav1.2 (pS1928); <b>Bottom</b>: results obtained with the anti-GAPDH antibody. Both Western blots shown are derived from the same membrane.</p>
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<p>Expression of Cav1.2 and baseline phosphorylation at S1928 in LV myocardium from <span class="html-italic">Cacna1c<sup>+/−</sup></span> and WT rats. (<b>A</b>) Original Western blot images of 8 WT (+/+) and 8 <span class="html-italic">Cacna1c<sup>+/−</sup></span> (+/−) samples derived from two membranes probed with the anti-Cav1.2 antibody. The protein used for normalization (GAPDH) derived from the same membranes is shown below the protein of interest. (<b>B</b>) The expression of Cav1.2 (normalized to GAPDH; Cav1.2/GAPDH) is decreased by ≈30% in <span class="html-italic">Cacna1c<sup>+/−</sup></span> (+/−). (<b>C</b>) Original Western blot images of 8 WT (+/+) and 8 <span class="html-italic">Cacna1c<sup>+/−</sup></span> (+/−) samples derived from two membranes probed with the anti-pS1928 antibody. The protein used for normalization (GAPDH) derived from the same membranes is shown below. (<b>D</b>) Phosphorylation status of S1928 in <span class="html-italic">Cacna1c<sup>+/−</sup></span> (+/−) versus WT (+/+) control samples when normalized to GAPDH (pS1928/GAPDH). Phosphorylation of S1928 is increased by ≈50% in <span class="html-italic">Cacna1c<sup>+/−</sup></span> (+/−). (<b>E</b>) Phosphorylation status of S1928 in <span class="html-italic">Cacna1c<sup>+/−</sup></span> (+/−) versus WT (+/+) control samples when normalized to Cav1.2 expression (pS1928/Cav1.2). The phosphorylation of S1928 is increased by ≈110% in <span class="html-italic">Cacna1c<sup>+/−</sup></span> (+/−). Circles represent number of animals: N = 8 (WT); N = 8 (<span class="html-italic">Cacna1c<sup>+/−</sup></span>); Student’s <span class="html-italic">t</span>-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; 0.0001. Further information and all original Western blot images from this series are shown in <a href="#app1-ijms-25-13713" class="html-app">Supplementary Materials</a>.</p>
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<p>Isoprenaline-Induced Increase in Phosphorylation of Cav1.2 at S1928 in LV Myocardium from WT and <span class="html-italic">Cacna1c<sup>+/−</sup></span> rats. (<b>A</b>) Original Western blot images of 16 WT (+/+) and 16 <span class="html-italic">Cacna1c<sup>+/−</sup></span> (+/−) LV samples probed with anti-Cav1.2 antibody. The protein used for normalization (GAPDH) derived from the same membranes is shown below the protein of interest. Hearts were either left untreated (Ctrl) or treated with 100 nM isoprenaline (Iso). (<b>B</b>) The expression of Cav1.2 normalized to GAPDH (Cav1.2/GAPDH) was reduced in <span class="html-italic">Cacna1c<sup>+/−</sup></span> (+/−) LV samples. (<b>C</b>) Original Western blot images of 16 WT (+/+) and 16 <span class="html-italic">Cacna1c<sup>+/−</sup></span> (+/−) LV samples probed with anti-phospho-S1928 antibody. The protein used for normalization (GAPDH) derived from the same membranes is shown below the protein of interest. Hearts were either left untreated (Ctrl) or treated with 100 nM isoprenaline (Iso). Iso-treated samples exhibit larger phosphorylation of S1928. (<b>D</b>) Phosphorylation of S1928 normalized to GAPDH expression (pS1928/GAPDH) in the four groups: WT untreated control (+/+ Ctrl), WT treated with Iso (+/+ Iso), <span class="html-italic">Cacna1c<sup>+/−</sup></span> untreated control (+/− Ctrl), and <span class="html-italic">Cacna1c<sup>+/−</sup></span> treated with Iso (+/− Iso). N = 8 samples for each group. (<b>E</b>) Phosphorylation of S1928 normalized to Cav1.2 expression (pS1928/Cav1.2) in the four groups: WT untreated control (+/+ Ctrl), WT treated with Iso (+/+ Iso), <span class="html-italic">Cacna1c<sup>+/−</sup></span> untreated control (+/− Ctrl), and <span class="html-italic">Cacna1c<sup>+/−</sup></span> treated with Iso (+/− Iso). N = 8 samples for each group. Iso increased phosphorylation of S1928 and the Iso-induced increase was larger in WT than in <span class="html-italic">Cacna1c<sup>+/−</sup></span> LV samples. Student’s <span class="html-italic">t</span>-test (<b>B</b>) or one-way ANOVA (<b>D</b>,<b>E</b>) were used for comparison of groups. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001. All original Western blot images from this series are shown in <a href="#app1-ijms-25-13713" class="html-app">Supplementary Materials</a>.</p>
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<p>Proposed alterations of Ca<sup>2+</sup> handling in ventricular myocytes from <span class="html-italic">Cacna1c<sup>+/−</sup></span> rats under basal conditions. The scheme depicts part of a ventricular myocyte from a wildtype (<b>top</b>) and a <span class="html-italic">Cacna1c<sup>+/−</sup></span> rat (<b>bottom</b>) with T-tubule, surface sarcolemma and the sarcoplasmic reticulum and the location of major Ca<sup>2+</sup>-regulating proteins: Cav1.2, RyR2, NCX, SERCA, and PLB. The space between the T-tubule (with Cav1.2) and the junctional SR (with RyR2) is termed dyadic cleft. In ventricular myocytes from <span class="html-italic">Cacna1c<sup>+/−</sup></span> rats, the expression of Cav1.2 is reduced by 30%, whereas the expression of NCX and SERCA is elevated by +20% and +50%, respectively. β-adrenergic receptors (β-ARs) are not activated (grey), and cAMP levels in the bulk cytosol are very low. Hence, most serine (S) residues in Cav1.2, RyR2, and PLB, which are targets of PKA, are not phosphorylated, as indicated by the white circles attached to the respective proteins. In the dyadic cleft of <span class="html-italic">Cacna1c<sup>+/−</sup></span> myocytes, however, Cav1.2 and RyR2 exhibit increased phosphorylation (P) of S1928 and S2808, respectively, as indicated by the yellow circles. The increased PKA-dependent phosphorylation of Cav1.2 and RyR2 is presumably caused by locally elevated cAMP concentration in the immediate vicinity of the channels, as depicted by the blue cAMP symbols. Abbreviations: AC, adenylate cyclase; β-AR, β-adrenergic receptor; P, phosphorylated serine residue; S, serine residue (non-phosphorylated). Created in BioRender. Königstein, D. (2024) <a href="https://BioRender.com/a97h526" target="_blank">https://BioRender.com/a97h526</a> (accessed on 17 December 2024).</p>
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<p>Proposed alterations of Ca<sup>2+</sup> handling in ventricular myocytes from <span class="html-italic">Cacna1c<sup>+/−</sup></span> rats during sympathetic stress. The scheme depicts part of a ventricular myocyte from a wildtype (<b>top</b>) and a <span class="html-italic">Cacna1c<sup>+/−</sup></span> rat (<b>bottom</b>) with T-tubule, surface sarcolemma, and the sarcoplasmic reticulum and the location of major Ca<sup>2+</sup>-regulating proteins: Cav1.2, RyR2, NCX, SERCA, and PLB. The space between the T-tubule (with Cav1.2) and the junctional SR (with RyR2) is termed dyadic cleft. In ventricular myocytes from <span class="html-italic">Cacna1c<sup>+/−</sup></span> rats, the expression of Cav1.2 is reduced by 30%, whereas the expression of NCX and SERCA is elevated by +20% and +50%, respectively. During the stimulation of β-AR with isoprenaline (ISO), mimicking sympathetic stress, cAMP levels increase throughout the cytosol and PKA phosphorylates target serine residues in Cav1.2 (S1928), RyR2 (S2808), and PLB (S16) in both wildtype and <span class="html-italic">Cacna1c<sup>+/−</sup></span> myocytes. In wildtype myocytes, all available serine residues in Cav1.2 and RyR2 become phosphorylated by PKA (yellow circles). Note, however, that for <span class="html-italic">Cacna1c<sup>+/−</sup></span> myocytes, not all available target serines (S) in Cav1.2 and RyR2 become phosphorylated by PKA, indicating a diminished phosphorylation reserve of these two channels. Abbreviations: AC, adenylate cyclase; β-AR, β-adrenergic receptor; ISO, isoprenaline; P, phosphorylated serine residue; S, serine residue (non-phosphorylated). Created in BioRender. Königstein, D. (2024) <a href="https://BioRender.com/o38g639" target="_blank">https://BioRender.com/o38g639</a> (accessed on 17 December 2024).</p>
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19 pages, 3899 KiB  
Article
Melatonin Enhances the Low-Calcium Stress Tolerance by Regulating Brassinosteroids and Auxin Signals in Wax Gourd
by Jingjing Chang, Xuemei Zhu, Yixuan Lian, Jing Li, Xiao Chen, Zhao Song, Lei Chen, Dasen Xie and Baige Zhang
Antioxidants 2024, 13(12), 1580; https://doi.org/10.3390/antiox13121580 (registering DOI) - 22 Dec 2024
Abstract
In plants, calcium (Ca) serves as an essential nutrient and signaling molecule. Melatonin is a biologically active and multi-functional hormone that plays an important role in improving nutrient use efficiency. However, its involvement in plant responses to Ca deficiency remains largely unexplored. This [...] Read more.
In plants, calcium (Ca) serves as an essential nutrient and signaling molecule. Melatonin is a biologically active and multi-functional hormone that plays an important role in improving nutrient use efficiency. However, its involvement in plant responses to Ca deficiency remains largely unexplored. This study aimed to assess the effects of melatonin on Ca absorption, the antioxidant system, and root morphology under low-Ca (LCa) stress conditions, as well as to identify key regulatory factors and signaling pathways involved in these processes using transcriptome analysis. Under LCa conditions, wax gourd seedling exhibited significant decreases in Ca accumulation, showed inhibition of root growth, and demonstrated the occurrence of oxidative damage. However, melatonin application significantly enhanced Ca content in wax gourd seedlings, and it enhanced the absorption of Ca2+ in roots by upregulating Ca2+ channels and transport genes, including BhiCNGC17, BhiCNGC20, BhiECA1, BhiACA1, and BhiCAX1. Furthermore, the application of exogenous melatonin mitigated the root growth inhibition and oxidative damage caused by LCa stress. This was evidenced by increases in the root branch numbers, root tips, root surface area, and root volume, as well as enhanced root vitality and antioxidant enzyme activities, as well as decreases in the reactive oxygen species content in melatonin treated plants. Transcriptome results revealed that melatonin mainly modulated the brassinosteroids (BRs) and auxin signaling pathway, which play essential roles in root differentiation, elongation, and stress adaptation. Specifically, melatonin increased the active BR levels by upregulating BR6ox (a BR biosynthesis gene) and downregulating BAS1 (BR degradation genes), thereby affecting the BR signaling pathway. Additionally, melatonin reduced IAA levels but activated the auxin signaling pathway, indicating that melatonin could directly stimulate the auxin signaling pathway via an IAA-independent mechanism. This study provides new insights into the role of melatonin in nutrient stress adaptation, offering a promising and sustainable approach to improve nutrient use efficiency in wax gourd and other crops. Full article
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Figure 1
<p>Effects of varied Ca concentrations on the relative expression of <span class="html-italic">BhiCOMT1</span> (<b>A</b>) and endogenous melatonin levels (<b>B</b>) in wax gourd roots. Wax gourd seedlings at the three-leaf stage were transferred to nutrient solutions containing varying Ca concentrations at 0, 0.04, 0.4, 2, and 4 mM for 1 d. Data in figures represent the means ± standard deviation (SD) of three replicates. Different letters represent significant differences at <span class="html-italic">p</span> &lt; 0.05. <span class="html-italic">BhiCOMT1</span>: cafeic acid <span class="html-italic">O</span>-methyltransferase in wax gourd (<span class="html-italic">Benincasa hispida</span> (Thunb.) Cogn). FW: fresh weight.</p>
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<p>Selection of the optimal melatonin concentration to enhance Ca absorption under LCa stress. Wax gourd seedlings at three-leaf stage were subjected to a LCa nutrient solution at a concentration of 0.04 mM and treated with varying concentrations of melatonin (0, 0.15, 1.5, 15, and 30 μM) in the rhizosphere for 10 d. The root and shoot samples were collected for measuring Ca contents. Data in figures represent the means ± standard deviation (SD) of three replicates. Different letters represent significant differences at <span class="html-italic"> p </span> &lt; 0.05. DW: dry weight; CK: control.</p>
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<p>Effect of rhizospheric melatonin application on Ca dynamics in wax gourd roots under LCa stress. (<b>A</b>) Ca content of different forms in root; (<b>B</b>) proportion of different forms of Ca; (<b>C</b>) net Ca<sup>2+</sup> flux; (<b>D</b>) relative expression of genes associated with Ca channels and transport. CK: control; LCa: low-Ca treatment; LCa+MT: low-Ca treatment + 1.5 μM melatonin treatment; DW: dry weight; Ca-WS: water-soluble Ca; Ca-PT: Ca-pectinate; Ca-Pho/Car: Ca phosphate/carbonate; Ca-Ox: Ca oxalate; <span class="html-italic">BhiCNGC17</span>: <span class="html-italic">cyclic nucleotide gated channel 17</span>; <span class="html-italic">BhiCNGC20</span>: <span class="html-italic">cyclic nucleotide gated channel 20</span>; <span class="html-italic">BhiECA1</span>: <span class="html-italic">calcium-transporting ATPase 1</span>; <span class="html-italic">BhiACA1</span>: <span class="html-italic">autoinhibited Ca<sup>2+</sup>-ATPase 1</span>; <span class="html-italic">BhiCAX1</span>: <span class="html-italic">vacuolar cation/proton exchanger 1</span>. Different letters indicate significant differences among treatments at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Effects of rhizospheric melatonin application on antioxidant system in root under LCa stress. Wax gourd seedlings at three-leaf stage were transferred to a low-Ca nutrient solution with concentrations of 0.04 mM and treated with 1.5 M melatonin in the rhizosphere for 10 d. (<b>A</b>) Root phenotype. (<b>B</b>) Root vitality. (<b>C</b>) Oxidative stress and antioxidant enzyme activities in roots. Data are means of three replicates (±standard deviation—SD). Different letters represent significant differences at <span class="html-italic">p</span> &lt; 0.05. CK: control; LCa: low-Ca treatment; LCa+MT: low-Ca treatment + 1.5 μM melatonin treatment; FW: fresh weight.</p>
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<p>Analysis of differentially expressed genes induced by melatonin and/or LCa stress based on mRNA-seq. Wax gourd seedlings at three-leaf stage were transferred to a low-Ca nutrient solution with concentrations of 0.04 mM and the rhizosphere was treated with 1.5 M melatonin. Root samples were taken at 24 h for mRNA sequencing. (<b>A</b>) Principal component analysis (PCA); (<b>B</b>) the number of DEGs; (<b>C</b>) Venn diagram of DEGs; (<b>D</b>) KEGG pathway analysis of DEGs. The X and Y axis represent enrichment factor and −log<sub>10</sub>(Q-value), respectively. (<b>E</b>) GO terms of DEGs classified as biological, cellular, and molecular functions. The X and Y axis represent −log<sub>10</sub>(Q-value) and pathway names, respectively. The digits in the columns indicate the DEGs numbers for each pathway. CK: control; LCa: low-Ca treatment; LCa+MT: low-Ca treatment + 1.5 μM melatonin treatment.</p>
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<p>A heatmap of genes related to brassinosteroid biosynthesis (<b>A</b>) and signaling (<b>B</b>) pathway based on mRNA-Seq and RT-qPCR after rhizospheric melatonin application in LCa stress plants. The log2 fold-change values (Log<sub>2</sub>FC) for genes in LCa vs. CK, LCa+MT vs. CK, and LCa+MT vs. LCa comparisons were used to generate a heatmap. Detailed genetic information in the heatmap is present in <a href="#app1-antioxidants-13-01580" class="html-app">Supplementary Table S1</a>. Different letters represent significant differences at <span class="html-italic">p</span> &lt; 0.05. CK: control; LCa: low-Ca treatment; LCa+MT: low-Ca treatment + 1.5 μM melatonin treatment; FW: fresh weight. “→” represents promotion; “<span class="html-fig-inline" id="antioxidants-13-01580-i001"><img alt="Antioxidants 13 01580 i001" src="/antioxidants/antioxidants-13-01580/article_deploy/html/images/antioxidants-13-01580-i001.png"/></span>” represents inhibition; boxes represent genes; circles represent metabolites.</p>
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<p>A heatmap of genes related to auxin signaling and metabolism pathway based on mRNA-Seq and RT-qPCR after rhizospheric melatonin application in LCa stress plants. (<b>A</b>) Auxin signaling pathway; (<b>B</b>) Auxin-actived signaling pathway; (<b>C</b>) Genes related to auxin metabolism; (<b>D</b>) Methyl indole-3-acetate and indole-3-acetic acid contents. The log2 fold-change values (Log<sub>2</sub>FC) for genes in LCa vs. CK, LCa+MT vs. CK, and LCa+MT vs. LCa comparisons were used to generate the heatmap. Specific genetic information is present in <a href="#app1-antioxidants-13-01580" class="html-app">Supplementary Table S1</a>. Different letters represent significant differences at <span class="html-italic">p</span> &lt; 0.05. CK: control; LCa: low-Ca treatment; LCa+MT: low-Ca treatment + 1.5 μM melatonin treatment; FW: fresh weight. “→” represents promotion; “<span class="html-fig-inline" id="antioxidants-13-01580-i001"><img alt="Antioxidants 13 01580 i001" src="/antioxidants/antioxidants-13-01580/article_deploy/html/images/antioxidants-13-01580-i001.png"/></span>” represents inhibition; boxes represent genes; circles represent metabolites.</p>
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15 pages, 3559 KiB  
Article
Tolerance of the Marine Anammox Candidatus Scalindua to High Nitrate Concentrations: Implications for Recirculating Aquaculture Systems
by Jonathan Armand Charles Roques, Ebuka Unegbu, Naoki Fujii, Amélie Marqué, Federico Micolucci, Kristina Snuttan Sundell and Tomonori Kindaichi
Water 2024, 16(24), 3705; https://doi.org/10.3390/w16243705 (registering DOI) - 22 Dec 2024
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Abstract
Recirculating aquaculture systems (RAS) hold significant potential for sustainable aquaculture by providing a stable, controlled environment that supports optimal fish growth and welfare. In RAS, ammonium (NH4+) is biologically converted into nitrate (NO3) via nitrite (NO2 [...] Read more.
Recirculating aquaculture systems (RAS) hold significant potential for sustainable aquaculture by providing a stable, controlled environment that supports optimal fish growth and welfare. In RAS, ammonium (NH4+) is biologically converted into nitrate (NO3) via nitrite (NO2) by nitrifying bacteria. As a result, NO3 usually accumulates in RAS and must subsequently be removed through denitrification in full RAS, or by regular water exchanges in partial RAS. The marine anammox bacteria Candidatus Scalindua can directly convert toxic NH4+ and NO2 into harmless nitrogen gas (N2) and has previously been identified as a promising alternative to the complex denitrification process or unsustainable frequent water exchanges in marine RAS. In this study, we evaluated the impact of high NO3 levels typically encountered in RAS on the performance and abundance of Ca. Scalindua in a laboratory-scale bioreactor. The bacterial composition of the granules, including the relative abundance of key nitrogen-cycling taxa, was analyzed along with the functional profile (i.e., NH4+ and NO2 removal efficiencies). For this purpose, a bioreactor was inoculated and fed a synthetic feed, enriched in NH4+, NO2, minerals and trace elements until stabilization (Phase 1, 52 days). NO3 concentrations were then gradually increased to 400 mg·L−1 NO3-N (Phase 2, 52 days), after which the reactor was followed for another 262 days (Phase 3). The reactor maintained high removal efficiencies; 88.0 ± 8.6% for NH4+ and 97.4 ± 1.7% for NO2 in Phase 2, and 95.0 ± 6.5% for NH4+ and 98.6 ± 2.7% for NO2 in Phase 3. The relative abundance of Ca. Scalindua decreased from 22.7% to 10.2% by the end of Phase 3. This was likely due to slower growth of Ca. Scalindua compared to heterotrophic bacteria present in the granule, which could use NO3 as a nitrogen source. Fluorescence in situ hybridization confirmed the presence of a stable population of Ca. Scalindua, which maintained high and stable NH4+ and NO2 removal efficiencies. These findings support the potential of Ca. Scalindua as an alternative filtering technology in marine RAS. Future studies should investigate pilot-scale applications under real-world conditions. Full article
(This article belongs to the Special Issue Advanced Use of Anammox Process in Wastewater Treatment)
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<p>Schematic drawing of the up-flow column reactor.</p>
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<p>Concentration of NH<sub>4</sub><sup>+</sup>, NO<sub>2</sub><sup>−</sup> and NO<sub>3</sub><sup>−</sup> in the influent (‘Inf’, filled circles) and effluent (‘Eff’, open circles) throughout the experiment.</p>
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<p>Anammox performance in the reactor. (<b>A</b>) NH<sub>4</sub><sup>+</sup> (closed diamonds) and NO<sub>2</sub><sup>−</sup> (open triangles) removal efficiencies (%) and expected NO<sub>3</sub><sup>−</sup> concentrations (dotted lines and open circles). (<b>B</b>) Nitrogen loading and removal rates (filled and open circles, respectively). Purple arrows indicate biomass sampling on days 38, 139, 167 and 331.</p>
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<p>Microbial community composition at the end of Phase 1 (day 38), and after one, two and nine months of exposure to 400 mg·L<sup>−1</sup> NO<sub>3</sub><sup>−</sup>-N (days 139, 167 and 331, respectively). Analysis based on 16S rRNA gene amplicon sequencing. Red percentages correspond to the relative abundance of the marine anammox <span class="html-italic">Ca.</span> Scalindua.</p>
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<p>FISH micrographs of biomass collected from the reactor on days 38 (<b>A</b>), 139 (<b>B</b>), 167 (<b>C</b>) and 331 (<b>D</b>). FISH analysis utilized the Alexa Fluor 647-labelled EUB338mix probe (cyan) targeting all bacteria, while the Alexa Fluor 555-labelled Sca1129b probe (red) was used specifically for <span class="html-italic">Ca.</span> Scalindua. <span class="html-italic">Ca.</span> Scalindua appears magenta and other bacteria appear blue. Scale bars indicate 10 μm.</p>
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17 pages, 4749 KiB  
Article
Selective O2/N2 Separation Using Grazyne Membranes: A Computational Approach Combining Density Functional Theory and Molecular Dynamics
by Adrià Calzada, Francesc Viñes and Pablo Gamallo
Nanomaterials 2024, 14(24), 2053; https://doi.org/10.3390/nano14242053 (registering DOI) - 22 Dec 2024
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Abstract
The separation of oxygen (O2) and nitrogen (N2) from air is a process of utmost importance nowadays, as both species are vital for numerous fundamental processes essential for our development. Membranes designed for their selective molecule separation have become [...] Read more.
The separation of oxygen (O2) and nitrogen (N2) from air is a process of utmost importance nowadays, as both species are vital for numerous fundamental processes essential for our development. Membranes designed for their selective molecule separation have become the materials of choice for researchers, primarily due to their ease of use. The present study proposes grazynes, 2D carbon-based materials consisting of sp and sp2 C atoms, as suitable membranes for separating O2 and N2 from air. By combining static density functional theory (DFT) calculations with molecular dynamics (MD) simulations, we address this issue through a comprehensive examination of the thermodynamic, kinetic, and dynamic aspects of the molecular diffusions across the nano-engineered pores of grazynes. The studied grazyne structures have demonstrated the ability to physisorb both O2 and N2, preventing material saturation, with diffusion rates exceeding 1 s−1 across a temperature range of 100–500 K. Moreover, they exhibit a selectivity of ca. 2 towards O2 at 300 K. Indeed, MD simulations with equimolar mixtures of O2:N2 indicated a selectivity towards O2 in both grazynes with ca. twice as many O2 filtered molecules in the [1],[2]{2}-grazyne and with O2 representing ca. 88% of the filtered gas in the [1],[2]{(0,0),2}-grazyne. [1],[2]{2}-grazyne shows higher permeability for both molecules compared to the other grazyne, with O₂ demonstrating particularly enhanced diffusion capacity across both membranes. Further MD simulations incorporating CO2 and Ar confirm O2 enrichment, particularly with [1],[2]{(0,0),2}-grazyne, which increased the presence of O2 in the filtered mixture by 26% with no evidence of CO2 molecules. Full article
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Figure 1
<p>Top view of [1],[2]{2}-grazyne. Dark and light green spheres correspond to <span class="html-italic">sp-</span> and <span class="html-italic">sp</span><sup>2</sup>-C atoms, respectively, while white spheres denote H atoms. The red lines fence the unit cell.</p>
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<p>Top view of [1],[2]{(0,0),2}-grazyne. Color code as in <a href="#nanomaterials-14-02053-f001" class="html-fig">Figure 1</a>.</p>
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<p>Diffusion paths for O<sub>2</sub> (left) and N<sub>2</sub> (right) across both grazyne membranes for the perpendicular (<span class="html-italic">prp</span>) and parallel (<span class="html-italic">pll</span>) conformations. TS represents the diffusion transition states and O<sub>2</sub>* and N<sub>2</sub>* the absorbed states of both molecules on the grazyne. Note that in the case of O<sub>2</sub> and the [1],[2]{2}-grazyne, the perpendicular and parallel diffusion energy paths are essentially coincidental and cannot be distinguished.</p>
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<p>Side (<b>top</b>) and top (<b>bottom</b>) views of the TSs corresponding to the diffusion process of N<sub>2</sub> (<b>left</b>) and O<sub>2</sub> (<b>right</b>) across the [1],[2]{2}-grazyne (upper images) or the [1],[2]{(0,0),2}-grazyne (lower images). Blue spheres denote N atoms while red ones denote O atoms. The rest of the code coloring is as in <a href="#nanomaterials-14-02053-f001" class="html-fig">Figure 1</a>.</p>
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<p>Side (<b>top</b>) and top (<b>bottom</b>) views of the N<sub>2</sub> (<b>left</b>) and O<sub>2</sub> (<b>right</b>) molecules adsorbed states on the [1],[2]{2}-grazyne (upper images) and the [1],[2]{(0,0),2}-grazyne (lower images). Coloring code as in <a href="#nanomaterials-14-02053-f002" class="html-fig">Figure 2</a>.</p>
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<p>Electrostatic potential maps for [1],[2]{2}-grazyne (<b>left</b>) and [1],[2]{(0,0),2}-grazyne (<b>right</b>) pores.</p>
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<p>Rate constants, <math display="inline"><semantics> <mrow> <msub> <mrow> <mi>r</mi> </mrow> <mrow> <mi>i</mi> </mrow> </msub> </mrow> </semantics></math>, for O<sub>2</sub> and N<sub>2</sub> diffusion across the grazyne membranes as a function of temperature.</p>
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<p>O<sub>2</sub> selectivity over N<sub>2</sub> for the studied grazynes in the range of 100–500 K.</p>
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<p>KPD of [1],[2]{2}-grazyne (black for N2 and green for O2) and [1],[2]{(0,0),2}-grazyne (blue for N<sub>2</sub> and red for O<sub>2</sub>) as a function of gas pressure and temperature. White regions denote preference for desorption (DES), whereas faded ones go for adsorbed situations (ADS). The grey dotted line indicates a total pressure of <span class="html-italic">p</span> = 1 atm and <span class="html-italic">T</span> = 300 K.</p>
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<p>Number of O<sub>2</sub> and N<sub>2</sub> molecules permeated across [1],[2]{2}-grazyne (<b>left</b>) and [1],[2]{(0,0),2}-grazyne (<b>right</b>) membranes as a function of simulation time, at <span class="html-italic">T</span> = 300 K, and at different gas pressures.</p>
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<p>Permeability at different gas stream pressures for O<sub>2</sub> or N<sub>2</sub> on both studied grazyne membranes and at <span class="html-italic">T</span> = 300 K. The permeability values are calculated from the average of multiple simulation replicates, with associated error bars representing the variability of the results.</p>
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24 pages, 3128 KiB  
Article
Seasonal and Spatial Discrimination of Sandy Beaches Using Energy-Dispersive X-Ray Fluorescence Spectroscopy Analysis: A Comparative Study of Maltese Bays
by Christine Costa, Frederick Lia and Emmanuel Sinagra
Environments 2024, 11(12), 299; https://doi.org/10.3390/environments11120299 (registering DOI) - 22 Dec 2024
Viewed by 10
Abstract
The general increase in awareness of environmental pollutants and typical sources reflects the application of sustainability and development goals. Energy-Dispersive X-Ray Fluorescence spectroscopy analysis has been used to analyse sand samples collected from five different beaches located on the east and north-eastern coasts [...] Read more.
The general increase in awareness of environmental pollutants and typical sources reflects the application of sustainability and development goals. Energy-Dispersive X-Ray Fluorescence spectroscopy analysis has been used to analyse sand samples collected from five different beaches located on the east and north-eastern coasts of Malta and Gozo during two summers and two winters. Samples were collected along linear transects perpendicular to the shoreline at three different depths. Chemometrics were used to discriminate between four latent variables, including season, location, depth, and distance from shoreline. The highest concentrations were attributed to Fe2O3, Al2O3, SrO, and SnO2. Principal Components Analysis and Factor Analysis classified distributions of Fe2O3, CoO, As2O3, MnO, SrO, SeO2, and CaCO3 under Principal Component 1. However, since no loading value dominance was observed, such distributions most likely represent a combination of lithogenic and anthropogenic natures. Discrimination using Stepwise Linear Canonical Discriminant Analysis (SLC-DA) and Partial Least Squares Discriminant Analysis (PLS-DA) using Leave-One-Out-Cross-Validation with Variance Importance Plots proved highly effective in classifying data by location, followed by seasonal variability. It follows that concentrations are not affected by depth and distance from shoreline variability, proving that accumulation and anthropogenic effects from land are not concentrated in specific zones but are spatially spread out along the bays and do not increase with depth. Full article
19 pages, 22817 KiB  
Article
Urban Single Precipitation Events: A Key for Characterizing Sources of Air Contaminants and the Dynamics of Atmospheric Chemistry Exchanges
by Maciej Górka, Aldona Pilarz, Magdalena Modelska, Anetta Drzeniecka-Osiadacz, Anna Potysz and David Widory
Water 2024, 16(24), 3701; https://doi.org/10.3390/w16243701 (registering DOI) - 22 Dec 2024
Viewed by 100
Abstract
The chemistry of atmospheric precipitation serves as an important proxy for discriminating the source(s) of air contaminants in urban environments as well as to discuss the dynamic of atmospheric chemistry exchanges. This approach can be undertaken at time scales varying from single events [...] Read more.
The chemistry of atmospheric precipitation serves as an important proxy for discriminating the source(s) of air contaminants in urban environments as well as to discuss the dynamic of atmospheric chemistry exchanges. This approach can be undertaken at time scales varying from single events to seasonal and yearly time frames. Here, we characterized the chemical composition of two single rain episodes (18 July 2018 and 21 February 2019) collected in Wrocław (SW Poland). Our results demonstrated inner variations and seasonality (within the rain event as well as between summer and winter), both in ion concentrations as well as in their potential relations with local air contaminants and scavenging processes. Coupling statistical analysis of chemical parameters with meteorological/synoptic conditions and HYSPLIT back trajectories allowed us to identify three main factors (i.e., principal components; PC) controlling the chemical composition of precipitation, and that these fluctuated during each event: (i) PC1 (40%) was interpreted as reflecting the long-range transport and/or anthropogenic influences of emission sources that included biomass burning, fossil fuel combustion, industrial processes, and inputs of crustal origin; (ii) PC2 (20%) represents the dissolution of atmospheric CO2 and HF into ionic forms; and (iii) PC3 (20%) originates from agricultural activities and/or biomass burning. Time variations during the rain events showed that each factor was more important at the start of the event. The study of both SO42− and Ca2+ concentrations showed that while sea spray inputs fluctuated during both rain events, their overall impact was relatively low. Finally, below-cloud particle scavenging processes were only observed for PM10 at the start of the winter rain episode, which was probably explained by the corresponding low rain intensity and an overlap from local aerosol emissions. Our study demonstrates the importance of multi-time scale approaches to explain the chemical variability in rainwater and both its relation to emission sources and the atmosphere operating processes. Full article
(This article belongs to the Section Urban Water Management)
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<p>Study sites in Wrocław (SW Poland): University of Wrocław (UWr), where precipitation was collected; IMWM and CIEP air quality monitoring stations.</p>
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<p>Time variations in the meteorological parameters and chemical composition for precipitation samples collected on 18 July 2018: (<b>A</b>) precipitation at IMWM station, wind velocity and air temperature at UWr station, wind rose (24 h); (<b>B</b>) SO<sub>2</sub>, NO<sub>x</sub>, PM<sub>10</sub>, PM<sub>2.5</sub>, O<sub>3</sub> concentrations at CIEP station; (<b>C</b>) anion concentrations in precipitation; (<b>D</b>) pH, EC, and cation concentrations in precipitation.</p>
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<p>Time variations in the meteorological parameters and chemical composition for precipitation samples collected during on 21 February 2019: (<b>A</b>) precipitation at IMWM and UWr stations, wind velocity and air temperature at UWr station, wind rose (24 h); (<b>B</b>) SO<sub>2</sub>, NO<sub>x</sub>, PM<sub>10</sub>, PM<sub>2.5</sub>, O<sub>3</sub> concentrations at CIEP station; (<b>C</b>) anion concentrations in precipitation; (<b>D</b>) pH, EC, and cation concentrations in precipitation.</p>
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<p>The 48 h NOAA HYSPLIT back trajectories showing air mass movement to Wrocław for the (<b>A</b>) summer (18 July 2018) and (<b>C</b>) winter (21 February 2019) precipitation episodes at 12:00 UTC. KNMI synoptic charts (<a href="https://www.knmi.nl" target="_blank">https://www.knmi.nl</a>, accessed on 29 March 2023) corresponding to the two SOM-based weather patterns at 12:00 UTC on (<b>B</b>) 18 July 2018 and (<b>D</b>) 21 February 2021. Prominent synoptic features: L—low-pressure system; H—high-pressure system; blue—cold front; red—warm front; magenta—occluded front.</p>
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<p>Time variations in the calculated concentrations of nSS and SS sulfates and nSS and SS calcium ions in rainwater for the (<b>A</b>,<b>B</b>) summer (18 July 2018) and (<b>C</b>,<b>D</b>) winter (21 February 2019) rain episodes. Equations used for calculations are detailed in the text.</p>
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<p>Time variations in the rainwater sample scores on each principal component analysis (PCA) principal component for (<b>A</b>) summer (18 July 2018) and (<b>B</b>) winter (21 February 2019) precipitation episodes. Results of the PCA for each precipitation event are also presented. Highlighted red values identify significant loadings.</p>
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13 pages, 1261 KiB  
Article
The Long-Term Effect of Cattle Manure Application on Soil P Availability and P Fractions in Saline-Sodic Soils in the Songnen Plain of China
by Xiaotong Feng, Changjie Liu, Yang Li, Jiaqi Xu, Juan Zhang and Qingfeng Meng
Agronomy 2024, 14(12), 3059; https://doi.org/10.3390/agronomy14123059 (registering DOI) - 22 Dec 2024
Viewed by 85
Abstract
Lower soil phosphorus (P) availability in saline-sodic soils is due to high pH and salinity, which seriously limited crop growth. Manure application has a positive effect on soil properties and P availability. We conducted an experiment, which included five treatments with different durations [...] Read more.
Lower soil phosphorus (P) availability in saline-sodic soils is due to high pH and salinity, which seriously limited crop growth. Manure application has a positive effect on soil properties and P availability. We conducted an experiment, which included five treatments with different durations of manure application: 11-, 16-, 22-, and 27-year manure treatments, and no manure as a control treatment (CK). The results showed that manure application decreased soil pH and electrical conductivity (EC) and increased soil organic matter (SOM). Soil available P content increased by 236.76 mg·kg−1 after applying manure for 27 years. Compared to the CK treatment, manure application significantly increased alkaline phosphatase (ALP) 3.36–6.05-fold and increased microbial biomass phosphorus (MBP) 3.69–15.90-fold (p < 0.05). The organic P (Po) and inorganic P (Pi) contents increased with manure application, except Ca10-P and O-P. Furthermore, we found that pH and EC were significantly negatively correlated with SOM (p < 0.05). MBP and ALP were significantly positively correlated with SOM (p < 0.05). Available P was mainly affected by Ca2-P (+0.71, p < 0.001). Overall, manure application in saline-sodic soils altered soil saline-sodic properties by increasing SOM. The results also indicated that enhanced soil available P is due to an increase in Ca2-P, Al-P, and Po mineralization, especially for Ca2-P. Full article
(This article belongs to the Section Soil and Plant Nutrition)
17 pages, 20035 KiB  
Article
Comparative Analysis of Ca2+/Cation Antiporter Gene Family in Rosa roxburghii and Enhanced Calcium Stress Tolerance via Heterologous Expression of RrCAX1a in Tobacco
by Tuo Zeng, Liyong Zhu, Wenwen Su, Lei Gu, Hongcheng Wang, Xuye Du, Bin Zhu, Caiyun Wang and Di Wu
Plants 2024, 13(24), 3582; https://doi.org/10.3390/plants13243582 (registering DOI) - 22 Dec 2024
Viewed by 105
Abstract
Rosa roxburghii, a calciphilic species native to the mountainous regions of Southwest China, is renowned for its high vitamin C and bioactive components, making it valuable for culinary and medicinal uses. This species exhibits remarkable tolerance to the high-calcium conditions typical of [...] Read more.
Rosa roxburghii, a calciphilic species native to the mountainous regions of Southwest China, is renowned for its high vitamin C and bioactive components, making it valuable for culinary and medicinal uses. This species exhibits remarkable tolerance to the high-calcium conditions typical of karst terrains. However, the underlying mechanisms of this calcium resilience remain unclear. The Ca2+/cation antiporter (CaCA) superfamily plays a vital role in the transport of Ca2+ and other cations and is crucial for plant tolerance to metal stress. However, the roles and evolutionary significance of the CaCA superfamily members in R. roxburghii remain poorly understood. This study identified 22 CaCA superfamily genes in R. roxburghii, categorized into four subfamilies. The gene structures of these RrCaCAs show considerable conservation across related species. Selection pressure analysis revealed that all RrCaCAs are subject to purifying selection. The promoter regions of these genes contain numerous hormone-responsive and stress-related elements. qRT-PCR analyses demonstrated that H+/cation exchanger (CAX) RrCAX1a and RrCAX3a were highly responsive to Ca2+ stress, cation/Ca2+ exchanger (CCX) RrCCX4 to Mg2+ stress, and RrCCX11a to Na+ stress. Subcellular localization indicated that RrCAX1a is localized to the plant cell membrane, and its stable transformation in tobacco confirmed its ability to confer enhanced resistance to heavy Ca2+ stresses, highlighting its crucial role in the high-calcium tolerance mechanisms of R. roxburghii. This research establishes a foundation for further molecular-level functional analyses of the adaptation mechanisms of R. roxburghii to high-calcium environments. Full article
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<p>Phylogenetic tree of <span class="html-italic">CaCA</span> gene family. Constructed using the maximum likelihood (ML) method with 1000 bootstrap replications, the tree classifies 22 <span class="html-italic">CaCA</span> genes from <span class="html-italic">R. roxburghii</span>, 12 from <span class="html-italic">A. thaliana</span>, and 19 from <span class="html-italic">V. duclouxii</span> into 4 subfamilies.</p>
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<p>Chromosomal localization of <span class="html-italic">RrCaCA</span> genes. The chromosomal locations of the <span class="html-italic">RrCaCA</span> genes are depicted on Chr 1, Chr 2, Chr 6, and Chr 7. Genes are marked in red text to indicate their positions, with physical positions scaled by megabases (Mb) shown on the left. Chromosomal background shading represents gene density, where red indicates high gene density regions and blue indicates low gene density regions.</p>
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<p>Structural motifs of <span class="html-italic">RrCaCA</span> genes. (<b>a</b>) MEME motifs; (<b>b</b>) conserved functional domains; (<b>c</b>) intron and exon structures.</p>
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<p>Collinearity analysis of <span class="html-italic">CaCA</span> genes. (<b>a</b>) Collinearity analysis of <span class="html-italic">RrCaCA</span> genes within <span class="html-italic">R. roxburghii</span> genome; (<b>b</b>) inter-genomic collinearity analysis among <span class="html-italic">A. thaliana</span>, <span class="html-italic">R. chinensis</span>, <span class="html-italic">R. wichuraiana</span>, and <span class="html-italic">R. rugosa</span>. Black and blue lines represent collinearity of <span class="html-italic">CaCA</span> genes.</p>
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<p>Ka/Ks values of <span class="html-italic">CaCAs</span> from four <span class="html-italic">Rosa</span> species: <span class="html-italic">R. roxburghii</span> (<span class="html-italic">Rro</span>), <span class="html-italic">R. chinensis</span> (<span class="html-italic">Rch</span>), <span class="html-italic">R. wichuraiana</span> (<span class="html-italic">Rwi</span>), and <span class="html-italic">R. rugosa</span> (<span class="html-italic">Rru</span>).</p>
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<p>Heatmap showing predicted cis-acting elements in promoter regions of <span class="html-italic">RrCaCA</span>. The bar chart on the left visualizes promoter elements, with colors representing functional categories as indicated in the legend. The heatmap on the right shows the frequency of specific cis-regulatory motifs in the promoters of the analyzed genes, with numbers indicating motif occurrences.</p>
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<p>Expression profiles of <span class="html-italic">RrCaCA</span> under metal stress conditions. The qRT-PCR results normalized with the 2<sup>−ΔΔCT</sup> Ct method using GADPH as an internal reference. The experiment was performed three times with three biological replicates. Error bars represent mean ± SD. CK refers to the untreated <span class="html-italic">R. roxburghii</span>, while the other treatments include <span class="html-italic">R. roxburghii</span> subjected to 200 mM NaCl, 50 mM MgCl<sub>2</sub>, 50 mM MnCl<sub>2</sub>, and 100 mM CaCl<sub>2</sub>. Different lowercase letters indicate significant differences, as determined by one-way ANOVA followed by Tukey’s post hoc test (<span class="html-italic">p</span> &lt; 0.05). Groups with different letters are significantly different from each other, with the letter assignments based on the results of pairwise comparisons.</p>
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<p>Subcellular localization of <span class="html-italic">RrCAX1a</span> and its role in enhancing calcium stress resistance in tobacco. (<b>a</b>) Subcellular localization of <span class="html-italic">RrCAX1a</span> showing its membrane-bound localization; CK represents control group transformed with empty pSuper1300-GFP vector. RrCAX1a-GFP indicates experimental group transformed with RrCAX1a-GFP fusion protein, demonstrating clear GFP signals localized to plasma membrane. GFP signals (<b>left</b>), bright-field images (<b>middle</b>), and merged images (<b>right</b>) are shown. Scale bar = 30 μm. (<b>b</b>) Overexpression of <span class="html-italic">RrCAX1a</span> in tobacco enhances resistance to calcium stress; CK represents transgenic tobacco plants transformed with pBI121 empty vector. OE represents transgenic tobacco plants transformed with pBI121-<span class="html-italic">RrCAX1a</span> vector. Scale bar = 2 cm.</p>
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15 pages, 1953 KiB  
Article
Analysis of Immunosuppression and Antioxidant Damage in Diploid and Triploid Crucian Carp (Carassius auratus) Induced by Saline-Alkaline Environmental Stress: From Metabolomic Insight
by Fangying Yuan, Xiaofeng Wei, Dongping Li, Xiaofeng Jin, Jing Wang and Yanchun Sun
Metabolites 2024, 14(12), 721; https://doi.org/10.3390/metabo14120721 (registering DOI) - 21 Dec 2024
Viewed by 255
Abstract
Objectives: The salinization of the water environment worldwide is increasing, which has brought great challenges to the sustainability of fish farming of aquatic animals. Methods: Three NaHCO3 concentration groups (0 mmol/L, 20 mmol/L, and 60 mmol/L) were set up in this study [...] Read more.
Objectives: The salinization of the water environment worldwide is increasing, which has brought great challenges to the sustainability of fish farming of aquatic animals. Methods: Three NaHCO3 concentration groups (0 mmol/L, 20 mmol/L, and 60 mmol/L) were set up in this study to investigate growth and metabolic differences between diploid and triploid crucian carp under saline-alkaline stresses. Purpose: This study utilized UPLC-QTOF/MS metabolomics to analyze significant metabolites and metabolic pathways in the serum of diploid and triploid crucian carp, exposing them to different NaHCO3 concentrations in saline-alkaline habitats, elucidating the mechanism of their metabolic differences. Results: Results revealed that in the CA20 group, diploid and triploid crucian carp shared 69 differential metabolites, primarily enriched in pathways such as sphingolipid metabolism, glycerophospholipid metabolism, and linoleic acid metabolism. In the CA60 group, 46 differentially metabolites (DMs) were identified, mainly enriched in pathways such as linoleic acid metabolism, unsaturated fatty acid biosynthesis and sphingolipid metabolism. Conclusions: The analysis indicated that under different carbonate-saline-alkaline concentrations, diploid and triploid crucian carp primarily enriched in metabolic pathways such as glycerophospholipid metabolism, sphingolipid metabolism, and unsaturated fatty acid biosynthesis. With increasing carbonate-alkaline concentrations, hemolytic phospholipids associated with cell apoptosis were significantly upregulated and sphingolipid metabolism related to inflammation was more significantly enriched in triploid crucian carp, indicating that triploid crucian carp exhibited significant sensitivity to high carbonate-saline-alkaline stress and poorer carbonate-saline-alkaline tolerance. The results of this study provided a scientific theoretical basis for the later cultivation and aquaculture research of saline-alkaline-tolerant fish species. Full article
(This article belongs to the Special Issue Metabolic Physiology Under Environmental Coercion)
21 pages, 19072 KiB  
Article
Early-Stage IM Treatment with the Host-Derived Immunostimulant CPDI-02 Increases Curative Protection of Healthy Outbred Mice Against Subcutaneous Infection with Community-Acquired Methicillin-Resistant Staphylococcus aureus USA300
by Jason P. Stewart, Caleb M. Sandall, Jacob E. Parriott, Stephen M. Curran, Russell J. McCulloh, Donald R. Ronning, Joy A. Phillips, Robin Schroeder, Christy Neel, Kelly F. Lechtenberg, Samuel M. Cohen, Yazen Alnouti, Sohel Daria, D. David Smith and Joseph A. Vetro
Pharmaceutics 2024, 16(12), 1621; https://doi.org/10.3390/pharmaceutics16121621 (registering DOI) - 21 Dec 2024
Viewed by 433
Abstract
Background/Objectives: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) greatly complicates the treatment of skin and soft tissue infections (SSTI). It was previously found that subcutaneous (SQ) treatment with the mononuclear phagocyte (MP)-selective activator complements peptide-derived immunostimulant-02 (CPDI-02; formerly EP67) and increases prophylaxis of outbred [...] Read more.
Background/Objectives: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) greatly complicates the treatment of skin and soft tissue infections (SSTI). It was previously found that subcutaneous (SQ) treatment with the mononuclear phagocyte (MP)-selective activator complements peptide-derived immunostimulant-02 (CPDI-02; formerly EP67) and increases prophylaxis of outbred CD-1 mice against SQ infection with CA-MRSA. Here, we determined if treatment with CPDI-02 also increases curative protection. Methods: Female CD-1 mice were challenged SQ with CA-MRSA USA300 LAC, then CPDI-02 or inactive scCPDI-02 was administered by a topical, SQ, IM, or IV route at 6 or 24 h post-challenge. Abscess sizes were compared over 10 days and CA-MRSA burden, neutrophils, MP, and pro-inflammatory cytokines were compared in subcutaneous abscesses. CPDI-02 PK and distribution in female CD-1 mice were compared after IM or IV dosing and CPDI-02 toxicity in male and female CD-1 mice was determined by IM dose escalation and repeat IM dosing. Results: Repeat IM treatment starting at 6 h post-challenge decreased maximum abscess surface area, CA-MRSA burden, and time to resolution, whereas repeat treatment by a topical, SQ, or IV route had no effect. Repeat treatment starting at 24 h post-challenge was ineffective by the current routes. Single IM treatment starting at 6 h post-challenge was as effective as repeat IM treatment, increased systemic exposure to CPDI-02, and, in subcutaneous abscesses, initially decreased IL-1β and increased MP. CPDI-02 was tolerated between 130 and 170 mg/kg after IM dose escalation and between 65 and 130 mg/kg after repeat IM dosing with males being more tolerant. Conclusions: Single early-stage IM treatment with CPDI-02 may increase curative protection against SSTI caused by CA-MRSA and/or other pathogens controlled by activated MP. Full article
(This article belongs to the Section Pharmacokinetics and Pharmacodynamics)
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<p>Repeat treatment with CPDI-02 increases curative protection of healthy female outbred mice against subcutaneous challenge with CA-MRSA depending on the route of administration. On Day 0, CA-MRSA (USA300 LAC, 5 × 10<sup>7</sup> CFU) was injected SQ from the left rear flank into the dorsal side of 6-week-old female outbred CD-1 mice. Vehicle alone (white circles) or vehicle containing the indicated dose of CPDI-02 (black circles) was administered (↑) at 6 h post-challenge, then daily over 5 days starting 24 h post-challenge by the (<b>A</b>) topical (directly onto abscess), (<b>B</b>) subcutaneous (left flank), (<b>C</b>) intramuscular (left caudal thigh muscle), or (<b>D</b>) intravenous (tail vein) route. Average abscess surface areas ± SEM (n = 10 mice) from vehicle alone or vehicle containing CPDI-02 were determined daily by quantitative image analysis (<a href="#app1-pharmaceutics-16-01621" class="html-app">Figure S1</a>) and compared for each administration route by repeated measurement two-way ANOVA with Geisser–Greenhouse correction.</p>
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<p>Single IM treatment with CPDI-02 is sufficient to increase curative protection of healthy female outbred mice against subcutaneous infection with CA-MRSA in a dose-dependent manner. On Day 0, CA-MRSA (USA300 strain, 5 × 10<sup>7</sup> CFU) was administered SQ from the left rear flank into the dorsal side of 6-week-old female outbred CD-1 mice. (<b>A</b>) At 6 h post-challenge, vehicle alone (PBS, white circles) or vehicle containing the indicated dose of CPDI-02 (black symbols) was administered IM (↑) to the left caudal thigh muscle. Average abscess surface areas ± SEM (n = 10 mice) were then determined daily starting 24 h post-challenge by quantitative image analysis (<a href="#app1-pharmaceutics-16-01621" class="html-app">Figure S1</a>) and compared between doses by repeated measurement two-way ANOVA with Geisser–Greenhouse correction and Tukey’s post-test. On Day 1 post-challenge, (<b>B</b>) average colony forming units (CFU) of CA-MRSA/g of abscess biopsy ± SD (n = 10 mice) after treatment with vehicle alone (white circles) or vehicle containing CPDI-02 at 50 mg/kg (black circles) were determined by dilution plating and compared by two-tailed Mann–Whitney test and (<b>C</b>) the presence of CA-MRSA in subcutaneous abscess cross-sections was determined by histochemistry (HC) with Gram staining and imaging at 40× (2 mm wide) and 200× (400 μm wide) magnification. HC images are representative of 5 mice from each treatment group.</p>
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<p>Single curative IM treatment with CPDI-02 increases the number of mononuclear phagocytes in subcutaneous abscesses from healthy female outbred mice at 18 h post-treatment. On Day 0, CA-MRSA (USA300 strain, 5 × 10<sup>7</sup> CFU) was administered SQ from the left rear flank into the dorsal side of 6-week-old healthy female outbred CD-1 mice. At 6 h post-challenge, vehicle alone (PBS) or vehicle containing CPDI-02 at 50 mg/kg was administered IM to the left caudal thigh muscle. On Day 1 post-challenge (18 h post-treatment), the presence of (<b>A</b>,<b>F</b>;<b>C</b>,<b>H</b>;<b>E</b>,<b>J</b>) cell nuclei (DAPI<sup>+</sup> cells, blue), (<b>B</b>,<b>G</b>;<b>C</b>,<b>H</b>) neutrophils (Ly6G/Ly6C<sup>+</sup> cells, red), and (<b>D</b>,<b>I</b>;<b>E</b>,<b>J</b>) mononuclear phagocytes (F4/80<sup>+</sup> cells, green) in subcutaneous abscesses was determined by IHC (200× magnification). Average ratios ±SD (n = 3 mice) of (<b>K</b>) neutrophils (Ly6G<sup>+</sup>/Ly6C<sup>+</sup>) to nuclei (DAPI<sup>+</sup>), (<b>L</b>) mononuclear phagocytes (F4/80<sup>+</sup>) to nuclei (DAPI<sup>+</sup>), and (<b>M</b>) mononuclear phagocytes (F4/80<sup>+</sup>) to neutrophils (Ly6G/Ly6C<sup>+</sup>) in subcutaneous abscesses were then determined by quantitative IHC image analysis and compared by unpaired t test with Welch’s correction. IHC images are representative of 3 mice from each treatment group.</p>
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<p>Single curative treatment with CPDI-02 by the IM route decreases early levels of IL-1β in subcutaneous abscesses of healthy female outbred mice after subcutaneous infection with CA-MRSA. On Day 0, CA-MRSA (USA300 strain, 5 × 10<sup>7</sup> CFU) was administered SQ from the left rear flank into the dorsal side of 6-week-old healthy female outbred CD-1 mice. At 6 h post-challenge, vehicle alone (PBS, white bars) or vehicle containing CPDI-02 at 50 mg/kg (black bars) was administered IM to the left caudal thigh muscle. Average concentrations of pro-inflammatory markers in subcutaneous abscess biopsies ±SD (n=5 mice per time point) at (<b>A</b>) 3 h post-treatment (9 h post-challenge) or (<b>B</b>) 18 h post-treatment (24 h post-challenge) were determined by multiplex ELISA and compared by two-tailed t test with Mann–Whitney post-test (P-values shown). No differences in other major murine cytokines/chemokines involved in inflammation (IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, CXCL-1 [KC/GRO], TNF-α) were observed at these time points (<a href="#app1-pharmaceutics-16-01621" class="html-app">Figure S3</a>).</p>
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<p>Preliminary PK and distribution of CPDI-02 in subcutaneous abscess-bearing female outbred mice after intravenous or intramuscular administration. On Day 0, CA-MRSA (USA300 strain, 5 × 10<sup>7</sup> CFU) was administered SQ from the left rear flank into the dorsal side of 6-week-old healthy female outbred CD-1 mice. At 6 h post-challenge, vehicle (PBS, white circles) or vehicle containing CPDI-02 (black circles) was injected into the tail vein [12.5 mg/kg] (IV) or left caudal thigh muscle [50 mg/kg] (IM) of healthy female outbred CD-1 mice (6 weeks old), then (<b>A</b>) average plasma concentrations of CPDI-02 ± SD (n = 5 mice) over 24 h post-treatment, and (<b>B</b>) average masses of CPDI-02/g of abscess or indicated organ ±SD (n = 3 to 5 mice) at 24 h post-challenge were determined by LC-MS/MS and compared by two-way ANOVA. AB—abscess; HT—heart; LG—lungs; LV—liver; KD—kidneys; SP—spleen; BR—brain.</p>
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<p>IM dose escalation of CPDI-02 in healthy male and female outbred CD-1 mice. (<b>A</b>) Vehicle alone (PBS, white symbols) or vehicle containing CPDI-02 (black symbols) was injected biweekly (↓) into the left caudal thigh muscle of healthy male and female outbred CD-1 mice (4 weeks old) at the indicated dose (black arrows) and average daily body weights ± SEM (n = 6 mice) were compared within each sex to vehicle alone by two-way ANOVA. (<b>B</b>) Pain and distress were scored on the day of each injection based on standardized signs in mice (<a href="#app1-pharmaceutics-16-01621" class="html-app">Table S1</a>). Signs of mild (score 2) to moderate (score 3) distress were observed starting 15 min after injection with CPDI-02 and resolved after 1 to 2 h. Four days after the final injection (Day 28), average values for complete blood count (CBC) with differential ±SD (n = 5 mice) were determined by hematology analyzer (<a href="#app1-pharmaceutics-16-01621" class="html-app">Table S2</a>) and compared by ordinary two-way ANOVA with uncorrected Fisher’s LSD and single pooled variance. Differences in CBC were only observed between (<b>C</b>) platelet concentrations and (<b>D</b>) platelet volumes.</p>
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<p>Repeat IM dosing of CPDI-02 in healthy male and female outbred mice. Vehicle alone (PBS) or vehicle containing CPDI-02 was injected biweekly (↓) into the left caudal muscle of healthy male and female outbred CD-1 mice (6 weeks old) at the indicated dose (black arrows) and (<b>A</b>) average daily body weights ± SEM (n = 10 mice) were compared within each sex to vehicle alone by two-way ANOVA and (<b>B</b>) survival for each treatment group was compared by simple Kaplan–Meier analysis with Mantel–Cox log-rank test (log-rank <span class="html-italic">p</span> values shown where relevant). No signs of distress were observed in any treatment group up to 2 h after injection. Four days after the final injection (Day 32), average values for complete blood count (CBC) with differential ±SD (<a href="#app1-pharmaceutics-16-01621" class="html-app">Table S3</a>) and analyte concentrations ±SD (<a href="#app1-pharmaceutics-16-01621" class="html-app">Table S4</a>) (n = 5 mice) were determined by a hematology and blood chemistry analyzer, respectively, and compared by ordinary two-way ANOVA with uncorrected Fisher’s LSD and single pooled variance. No differences in CBC were observed between (<b>C</b>) platelet concentrations and (<b>D</b>) platelet volumes.</p>
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