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Search Results (729)

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Keywords = morphometry

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11 pages, 1232 KiB  
Article
The Effects of Stocking Density and Food Deprivation on Mucous Cells and Lysozyme Activity in the Skin and Gills of Silver Catfish
by Gabriela Pires Scherer, Isadora Maria Oliveira Zavaglia, Fernando Jonas Sutili, Hugo Napoleão Pereira Silva, Magale Dallaporta Furquim, Marcelo Leite da Veiga, Bernardo Baldisserotto and Sílvio Teixeira da Costa
Animals 2023, 13(22), 3438; https://doi.org/10.3390/ani13223438 - 7 Nov 2023
Viewed by 1024
Abstract
This study aimed to identify the regions of the body surface of silver catfish (Rhamdia quelen) with a higher population of mucous cells in the skin. Additionally, the effects of stressful conditions, such as different stocking densities and food deprivation, on [...] Read more.
This study aimed to identify the regions of the body surface of silver catfish (Rhamdia quelen) with a higher population of mucous cells in the skin. Additionally, the effects of stressful conditions, such as different stocking densities and food deprivation, on the proliferative response of mucous cells in the skin and gill epithelium and their impact on cutaneous mucous lysozyme activity were investigated. Silver catfish were divided into four experimental groups: high stocking density (32 kg/m3) and fed (HSD-F), high stocking density and fasted (HSD-FS), low stocking density (2.5 kg/m3) and fed (LSD-F), and low stocking density and fasted (LSD-FS). Fish in the fed groups received commercial feed twice a day, amounting to 1% of the tank biomass. After a 14-day experimental period, the fish were anesthetized and euthanized. Samples of cutaneous mucous and skin fragments from seven different points and the second left branchial arch were collected. Histological slides of the skin and gills were stained with PAS + Alcian Blue at pH 2.5, and the epidermal mucous lysozyme activity was assessed using the turbidimetric method. The ventral point in front of the ventral fin was found to be the optimal location for collecting cutaneous epithelia due to its higher density of mucous cells. The population of mucous cells in both the skin and gills varied based on the collection point and treatment applied. The highest lysozyme activity in the epidermal mucous was observed in fish from the HSD-F group. Overall, these findings suggest that stocking density and food deprivation create stressful conditions for silver catfish, which modulate their mucosal response to each situation. Full article
(This article belongs to the Special Issue Feed Ingredients and Fish Mucosal Health)
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Figure 1

Figure 1
<p>Representation of collection sites for obtaining skin samples from silver catfish (<span class="html-italic">Rhamdia quelen</span>). DDF—Dorsofrontal to dorsal fin; CO—Caudal to operculum; VVF—Ventrofrontal to ventral fin; LVLB—Left ventrolateral to lateral band; LDSB—Left dorsolateral to side band; DCDF—Dorsum caudal to dorsal fin; VCVF—Ventrocaudal to ventral fin.</p>
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<p>Quantitative parameters from the basal group. (<b>A</b>) Density of goblet cells. (<b>B</b>) Subcutaneous dermal thickness. The numbers represent the collection sites of the skin samples from silver catfish (<span class="html-italic">Rhamdia quelen</span>). 1. CO—Caudal to operculum; 2. DCDF—Dorsum caudal to dorsal fin; 3. DDF—Dorsofrontal to dorsal fin; 4. LDSB—Left dorsolateral to side band; 5. VCVF—Ventrocaudal to ventral fin; 6. VVF—Ventrofrontal to ventral fin; 7. LVLB—Left ventrolateral to lateral band. N = 6–8, Kruskal–Wallis one way analysis of variance (ANOVA) on ranks (<span class="html-italic">p</span> ≤ 0.001).</p>
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<p>Cutaneous histology of silver catfish (<span class="html-italic">Rhamdia quelen</span>) under different stocking densities and feeding conditions. Photomicrographs of Alcian Blue-stained mucosal cells collected from the CO. (<b>A</b>) HSD-F: high stocking density (32 kg/m<sup>3</sup>) and fed; (<b>B</b>) HSD-FS: high stocking density and fasted; (<b>C</b>) LSD-F: low stoking density (2.5 kg/m<sup>3</sup>) and fed; and (<b>D</b>) LSD-FS: low stocking density and fasted (10× photomicrographs and 40 µm scale bar).</p>
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23 pages, 3667 KiB  
Review
History of Environmental and Climatic Changes Recorded in Lacustrine Sediments—A Wigry Lake Case Study
by Anna Kostka
Sustainability 2023, 15(21), 15628; https://doi.org/10.3390/su152115628 - 5 Nov 2023
Viewed by 1111
Abstract
Wigry Lake represents one of the most beautiful and valuable postglacial lakes that is located in the north-eastern part of Poland. It has been an object of scientific interest for over a century, but the most intense period of research started in 1997 [...] Read more.
Wigry Lake represents one of the most beautiful and valuable postglacial lakes that is located in the north-eastern part of Poland. It has been an object of scientific interest for over a century, but the most intense period of research started in 1997 and resulted in the production of numerous papers of a multidisciplinary range. The lake is especially well analyzed in terms of its sediments, which were studied using geophysical methods and using traditional lake cartography based on the sampling of sediments and their geochemical characterization. Nearly two decades of research has resulted in the extraction of over 1200 sediment samples, which facilitated the analysis and the description of five main sediment types that can be found at the bottom of Wigry Lake, i.e., carbonate gyttja, lacustrine chalk, clastic sediment, fluvial-lacustrine sediment, and organic gyttja. A very thorough vertical as well as spatial examination of Wigry Lake sediments, together with paleobiological research and isotopic dating, allowed researchers to analyze the history of environmental changes in the lake and its immediate vicinity, including anthropogenic changes. Wigry Lake had been preserved as a pristine lake for a long time, and, despite the significant growth of anthropopressure, which began in the 17th century and intensified in the 20th century, it retained its natural character to a large extent. This was possible mainly due to the favorable morphometry of the lake (large depth and capacity of the reservoir) and different forms of active and passive protection, introduced mainly after the establishment of the Wigry National Park in 1989. Full article
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Figure 1
<p>Study area: Wigry Lake within the borders of the Wigry National Park (WNP) (according to [<a href="#B64-sustainability-15-15628" class="html-bibr">64</a>], modified).</p>
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<p>Spatial characterization of the sediments of Wigry Lake: (<b>A</b>) a bathymetry sketch of the bottom (according to [<a href="#B102-sustainability-15-15628" class="html-bibr">102</a>]); (<b>B</b>) a map of the extent of individual facies and fragments of exemplary seismoacoustic cross-sections (according to [<a href="#B92-sustainability-15-15628" class="html-bibr">92</a>,<a href="#B103-sustainability-15-15628" class="html-bibr">103</a>], modified); (<b>C</b>) sampling map; (<b>D</b>) map of the extent of individual sediment types and other sedimentological phenomena (according to [<a href="#B89-sustainability-15-15628" class="html-bibr">89</a>,<a href="#B92-sustainability-15-15628" class="html-bibr">92</a>,<a href="#B96-sustainability-15-15628" class="html-bibr">96</a>,<a href="#B104-sustainability-15-15628" class="html-bibr">104</a>,<a href="#B105-sustainability-15-15628" class="html-bibr">105</a>] <span class="html-italic">vide</span> [<a href="#B42-sustainability-15-15628" class="html-bibr">42</a>], modified).</p>
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<p>Lithological characterization of the sediments of Wigry Lake: (<b>A</b>) examples of sediment core profiles of various lengths, from about 20 cm to 100 cm (according to [<a href="#B14-sustainability-15-15628" class="html-bibr">14</a>]) (from left to right: two organic gyttja cores; core consisting of lacustrine chalk in the bottom, turning gradually into carbonate gyttja in the upper part; three cores of lacustrine chalk enriched with organic matter in their upper parts); (<b>B</b>) upper part of carbonate gyttja with lamination; (<b>C</b>) calcareous tubes and clumps washed out from lacustrine chalk (according to [<a href="#B92-sustainability-15-15628" class="html-bibr">92</a>], photo: L. Krzysztofiak); (<b>D</b>) shell outwash (according to [<a href="#B92-sustainability-15-15628" class="html-bibr">92</a>], photo: J. Rutkowski).</p>
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16 pages, 7805 KiB  
Article
Vanilla pompona Schiede (Vanilloideae-Orchidaceae): Morphological Variation of the Labellum in the Mexican Localities of Veracruz, Puebla, Jalisco and Oaxaca
by Cecilia Viveros-Antonio, Adriana Delgado-Alvarado, Angel Bustamante-González, Jesús Hernández-Ruíz, Ma. de Lourdes Arévalo-Galarza and Braulio Edgar Herrera-Cabrera
Diversity 2023, 15(11), 1125; https://doi.org/10.3390/d15111125 - 31 Oct 2023
Viewed by 1436
Abstract
Vanilla pompona is the third most important commercial species of the genus Vanilla, but the morphometric variation of its flowers is not well known. In orchids, infraspecific variation is expressed in the labellum. This study had the objective of analyzing the floral [...] Read more.
Vanilla pompona is the third most important commercial species of the genus Vanilla, but the morphometric variation of its flowers is not well known. In orchids, infraspecific variation is expressed in the labellum. This study had the objective of analyzing the floral variation of V. pompona collections from the localities in Veracruz, Puebla, Jalisco and Oaxaca, in Mexico. During the flowering period, we obtained 55 collections of V. pompona flowers, and the labellum of each flower was dissected. We used ImageJ to measure and portray 54 lines and 7 angles in these labella. With the data obtained, we performed an analysis of variance, principal component analysis and cluster analysis. The results showed significant differences among the collections and localities in the means of the lines of the basal, middle, and apical regions of the labellum. Also, six morphotypes were obtained, and we found that variation was associated with factors such as the environment, geographic barriers, and pollinator pressure. Moreover, we observed variation in flowering periods in one locality of Veracruz. We concluded that there is infraspecific variation within and between the collection localities of V. pompona in the Atlantic region (Puebla and Veracruz) and the Pacific region (Jalisco and Oaxaca), Mexico. Full article
(This article belongs to the Special Issue Genetic Diversity, Ecology and Conservation of Endangered Species)
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Figure 1

Figure 1
<p>Geographical location of the collection sites of <span class="html-italic">Vanilla pompona</span> flowers in Mexico. The red dots show the collection sites: Cabo Corrientes (Jalisco), Santa Cruz Itundujia, Pluma Hidalgo and Santa María Chimalapa (Oaxaca), Tuzamapan de Galeana (Puebla), Papantla, Tihuatlán and Gutiérrez Zamora (Veracruz).</p>
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<p>Phenological stages of <span class="html-italic">Vanilla pompona</span>; flowering in April and mature fruits in December.</p>
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<p><span class="html-italic">Vanilla pompona</span> flower, labellum separated and dyed, fragmentation of the labellum into the (<b>a</b>) basal region, (<b>b</b>) middle region and (<b>c</b>) apical region.</p>
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<p>(<b>A</b>) Variables that define each of the first three principal components. (<b>B</b>) Dispersion of the 55 collections of the flower labellum of <span class="html-italic">Vanilla pompona</span> based on the first three principal components from the localities of Veracruz, Jalisco, Puebla and Oaxaca, Mexico. The colors in the labellum diagram correspond to the PCs, the blue color corresponds to the PC1 variables, yellow colors correspond to the PC2 variables, and the red color correspond to the PC3 variables. GI to GVI: Group I to Group IV. Each group had a different color to differentiate them.</p>
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<p>Dendrogram of the labellum morphotypes of <span class="html-italic">Vanilla pompona</span> from the localities of Veracruz, Jalisco, Puebla and Oaxaca, Mexico, based on the average of 61 variables and grouping by similarity distance. MI to MVI: Morphotype I to Morphotype IV. Each morphotype had a different color to differentiate them.</p>
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15 pages, 1579 KiB  
Article
A Novel Algorithm for Evaluating Bone Metastatic Potential of Breast Cancer through Morphometry and Computational Mathematics
by Simona-Alina Duca-Barbu, Alexandru Adrian Bratei, Antonia-Carmen Lisievici, Tiberiu Augustin Georgescu, Bianca Mihaela Nemes, Maria Sajin and Florinel Pop
Diagnostics 2023, 13(21), 3338; https://doi.org/10.3390/diagnostics13213338 - 30 Oct 2023
Viewed by 1108
Abstract
Bone metastases represent about 70% of breast cancer metastases and are associated with worse prognosis as the tumor cells acquire more aggressive features. The selection and investigation of patients with a high risk of developing bone metastasis would have a significant impact on [...] Read more.
Bone metastases represent about 70% of breast cancer metastases and are associated with worse prognosis as the tumor cells acquire more aggressive features. The selection and investigation of patients with a high risk of developing bone metastasis would have a significant impact on patients’ management and survival. The patients were selected from the database of Carol Davila Clinical Nephrology Hospital of Bucharest. Their tumor specimens were pathologically processed, and a representative area was selected. This area was scanned using an Olympus VS200 slide scanner and further analyzed using QuPath software v0.4.4. A representative group of approximately 60–100 tumor cells was selected from each section, for which the following parameters were analyzed: nuclear area, nuclear perimeter, long axis and cell surface. Starting from these measurements, the following were calculated: the mean nuclear area and mean nuclear volume, the nucleus to cytoplasm ratio, the length of the two axes, the long axis to short axis ratio, the acyclicity and anellipticity grade and the mean internuclear distance. The tumor cells belonging to patients known to have bone metastasis seemed to have a lower nuclear area (<55 µm2, p = 0.0035), smaller long axis (<9 µm, p = 0.0015), smaller values for the small axis (<7 µm, p = 0.0008), smaller mean nuclear volume (<200 µm3, p = 0.0146) and lower mean internuclear distance (<10.5 µm, p = 0.0007) but a higher nucleus to cytoplasm ratio (>1.1, p = 0.0418), higher axis ratio (>1.2, p = 0.088), higher acyclicity grade (>1.145, p = 0.0857) and higher anellipticity grade (>1.14, p = 0.1362). These parameters can be used for the evaluation of risk category of developing bone metastases. These results can be useful for the evaluation of bone metastatic potential of breast cancer and for the selection of high-risk patients whose molecular profiles would require further investigations and evaluation. Full article
(This article belongs to the Section Pathology and Molecular Diagnostics)
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<p>The measurements of nuclear dimensions and long axis. In first case (<b>left</b>), the cellular membrane cannot be identified on slides, so it was not measured, while, in the second case (<b>right</b>), the cellular membrane could be evaluated and measured.</p>
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<p>The evaluation and quantification of cytoplasm in tumor cells. In first case (<b>left</b>), the cellular membrane can be easily identified on slides, so it was not measured, while, in the second case (<b>right</b>), the cellular membrane could not be evaluated and measured, so areas including more cells were measured and the nuclei number was counted.</p>
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<p>The comparison of breast cancer patients by number of respected criteria.</p>
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23 pages, 6478 KiB  
Article
Experimental Early Stimulation of Bone Tissue Neo-Formation for Critical Size Elimination Defects in the Maxillofacial Region
by Nadezhda Nicolaevna Patlataya, Igor Nicolaevich Bolshakov, Anatoliy Alexandrovich Levenets, Nadezhda Nicolaevna Medvedeva, Vladimir Alexeevich Khorzhevskii and Mariya Arturovna Cherkashina
Polymers 2023, 15(21), 4232; https://doi.org/10.3390/polym15214232 - 26 Oct 2023
Cited by 1 | Viewed by 1034
Abstract
A biomaterial is proposed for closing extensive bone defects in the maxillofacial region. The composition of the biomaterial includes high-molecular chitosan, chondroitin sulfate, hyaluronate, heparin, alginate, and inorganic nanostructured hydroxyapatite. The purpose of this study is to demonstrate morphological and histological early signs [...] Read more.
A biomaterial is proposed for closing extensive bone defects in the maxillofacial region. The composition of the biomaterial includes high-molecular chitosan, chondroitin sulfate, hyaluronate, heparin, alginate, and inorganic nanostructured hydroxyapatite. The purpose of this study is to demonstrate morphological and histological early signs of reconstruction of a bone cavity of critical size. The studies were carried out on 84 white female rats weighing 200–250 g. The study group consisted of 84 animals in total, 40 in the experimental group and 44 in the control group. In all animals, three-walled bone defects measuring 0.5 × 0.4 × 0.5 cm3 were applied subperiosteally in the region of the angle of the lower jaw and filled in the experimental group using lyophilized gel mass of chitosan–alginate–hydroxyapatite (CH–SA–HA). In control animals, the bone cavities were filled with their own blood clots after bone trepanation and bleeding. The periods for monitoring bone regeneration were 3, 5, and 7 days and 2, 3, 4, 6, 8, and 10 weeks. The control of bone regeneration was carried out using multiple morphological and histological analyses. Results showed that the following process is an obligatory process and is accompanied by the binding and release of angiogenic implantation: the chitosan construct actively replaced early-stage defects with the formation of full-fledged new bone tissue compared to the control group. By the 7th day, morphological analysis showed that the formation of spongy bone tissue could be seen. After 2 weeks, there was a pronounced increase in bone volume (p < 0.01), and at 6 weeks after surgical intervention, the closure of the defect was 70–80%; after 8 weeks, it was 100% without violation of bone morphology with a high degree of mineralization. Thus, the use of modified chitosan after filling eliminates bone defects of critical size in the maxillofacial region, revealing early signs of bone regeneration, and serves as a promising material in reconstructive dentistry. Full article
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<p>Experimental study design.</p>
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<p>Regeneration of a bone defect in the lower jaw at the early stages of this experiment. (<b>A</b>)—experimental group; day 3 of this experiment, there is no bone tissue at the site of the defect; mesenchymal stromal cells (<b>^</b>) migrate to the area of the blood clot (<b>↑</b>) for further differentiation into osteoblasts; in the upper left corner of the control group, day 3 of this experiment, a formed defect with a blood clot (↑) is visible; the periodontal ligament (<b>*</b>) and the cortical plate of the lower jaw (<b>^</b>) are visible; (<b>B</b>)—CH–SA–HA group, day 7 of this experiment, islets of regenerating bone tissue (<b>↑</b>) in the area of hemorrhagic impregnation (<b>^</b>); (<b>C</b>)—control group, day 7 of this experiment, the bone defect is filled with immature connective tissue (<b>↑</b>).</p>
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<p>Changes in the area of the bone defect characterizing the active phase of bone tissue regeneration in healthy and experimental groups of rats: (<b>A</b>)—CH–SA–HA group, 2nd week of the experiment. On the surface of the bone beams (<b>*</b>), multiple osteoblasts are visible (<b>↑</b>), and a formed gap with an osteoclast is also visible (<span class="html-fig-inline" id="polymers-15-04232-i001"><img alt="Polymers 15 04232 i001" src="/polymers/polymers-15-04232/article_deploy/html/images/polymers-15-04232-i001.png"/></span>). Hematoxylin–eosin staining; (<b>B</b>)—control group, 2nd week of the experiment; the osteoblastic surface of the bone beams is significantly smaller in comparison with the experimental group (<b>↑</b>). Hematoxylin–eosin staining.</p>
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<p>(<b>A</b>)—The control group, 3rd week of the experiment, formed thin bone trabeculae in the area of the defect (<b>↑</b>) with pronounced intertrabecular spaces (<span class="html-fig-inline" id="polymers-15-04232-i002"><img alt="Polymers 15 04232 i002" src="/polymers/polymers-15-04232/article_deploy/html/images/polymers-15-04232-i002.png"/></span>); bone trabeculae are covered with cell mass (<b>^</b>); (<b>B</b>)—CH–SA–HA group, 4 weeks. Visible bone beams with predominantly free surfaces (↑) and intertrabecular spaces (<b>^</b>); the adjacent peripheral area with the growth of maturing connective tissue (<b>*</b>). Hematoxylin–eosin staining.</p>
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<p>The density distribution of the variable is “osteoblast surface”.</p>
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<p>The distribution density of the variable is “osteoid surface”.</p>
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<p>The distribution density of the variable is the “free surface” in the studied groups at different times of the experiment.</p>
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<p>Late terms of reorganization of the bone cavity in the experimental group. (<b>A</b>)—6 weeks, oriented bone beams with a mass of flattened osteoblasts (<span class="html-fig-inline" id="polymers-15-04232-i003"><img alt="Polymers 15 04232 i003" src="/polymers/polymers-15-04232/article_deploy/html/images/polymers-15-04232-i003.png"/></span>), adjacent peripheral area with proliferation of maturing connective tissue, active vascular endothelialization of bone regeneration, single osteoclasts (<b>↑</b>), organization of compact bone and periodontal ligament (<b>*</b>); (<b>B</b>)—8 weeks, large amount of spongy (<b>^</b>) and compact (<b>*</b>) bone tissue with cellular reaction on the bone beams (<b>↑</b>) and the formation of many micro-vessels; (<b>C</b>)—experimental group, 10 weeks, the mandibular defect is closed by a compact bone, in the border zone there is mature connective tissue (<b>↑</b>), in the adjacent bone tissue (<b>*</b>)—reactive bone marrow structures (<b>^</b>), formed osteons (*), many Haversian canals with a vascular component; Hematoxylin–eosin staining.</p>
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<p>The density of the distribution of the variable, “increase in bone volume”, in the study groups at randomly selected intervals of the experiment.</p>
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17 pages, 1751 KiB  
Article
Weak Genetic Isolation and Putative Phenotypic Selection in the Wild Carnation Dianthus virgineus (Caryophyllaceae)
by Jacopo Franzoni, Giovanni Astuti and Lorenzo Peruzzi
Biology 2023, 12(10), 1355; https://doi.org/10.3390/biology12101355 - 23 Oct 2023
Cited by 1 | Viewed by 2693
Abstract
By relating genetic divergence at neutral loci, phenotypic variation, and geographic and environmental distances, it is possible to dissect micro-evolutionary scenarios involving natural selection and neutral evolution. In this work, we tested the patterns of intraspecific genetic and phenotypic variation along an elevational [...] Read more.
By relating genetic divergence at neutral loci, phenotypic variation, and geographic and environmental distances, it is possible to dissect micro-evolutionary scenarios involving natural selection and neutral evolution. In this work, we tested the patterns of intraspecific genetic and phenotypic variation along an elevational gradient, using Dianthus virgineus as study system. We genotyped genome-wide SNPs through ddRAD sequencing and quantified phenotypic variation through multivariate morphological variation. We assessed patterns of variation by testing the statistical association between genetic, phenotypic, geographic, and elevational distances and explored the role of genetic drift and selection by comparing the Fst and Pst of morphometric traits. We revealed a weak genetic structure related to geographic distance among populations, but we excluded the predominant role of genetic drift acting on phenotypic traits. A high degree of phenotypic differentiation with respect to genetic divergence at neutral loci allowed us to hypothesize the effect of selection, putatively fuelled by changing conditions at different sites, on morphological traits. Thus, natural selection acting despite low genetic divergence at neutral loci can be hypothesized as a putative driver explaining the observed patterns of variation. Full article
(This article belongs to the Section Evolutionary Biology)
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Figure 1
<p>Patterns of overall geographic, phenotypic, and genetic variation among studied populations of <span class="html-italic">Dianthus virgineus</span>. (<b>a</b>) Study area and geographic distribution of the sampled populations, including elevations; (<b>b</b>) scatter plot of the first two components resulting from the Principal Component Analysis (PCA) on the refined morphometric dataset (for variable loading codes, see <a href="#biology-12-01355-t001" class="html-table">Table 1</a>; the variation in basal leaves length and calyx length along PC1 and the variation in plant height along PC2 are graphically represented); (<b>c</b>) major-mode barplots at K = 3 (highest ΔK) resulting from customized STRUCTURE analysis on the genetic matrix (populations are arranged according to increasing latitude, with the exception of EL and CAP).</p>
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<p>Variation with elevation and comparisons of phenotypic and neutral genetic differentiation of calyx length (<b>a</b>,<b>d</b>), plant height (<b>b</b>,<b>e</b>), and basal leaf width (<b>c</b>,<b>f</b>) in the studied populations of <span class="html-italic">Dianthus virgineus</span>; (<b>a</b>–<b>c</b>) relations of the three morphological features with elevation. Mean values (±SD) for each character is presented. Regression lines and adjusted R<sup>2</sup> are shown; (<b>d</b>–<b>f</b>) Pst-Fst comparisons for a range of c/h<sup>2</sup> values. Green lines represent the upper limit of 99% confidence intervals of Fst values. For Pst variation (red lines), 99% confidence intervals are shown as dashed lines.</p>
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<p>Relations between neutral genetic, phenotypic geographic, and elevational distance in <span class="html-italic">Dianthus virgineus</span>. Scatter plots of six Mantel tests are shown. Each point represents a pairwise comparison between two different populations. (<b>a</b>) Variation of pairwise genetic fixation of populations (GEN) with increasing geographic distance (GEO); (<b>b</b>) variation of GEN with increasing elevational distance (ELE); (<b>c</b>) variation of pairwise Euclidean distance of uncorrelated morphometric features (MOR) with ELE; (<b>d</b>) variation of MOR with ELE; (<b>e</b>) variation of MOR with GEN; (<b>f</b>) variation of ELE with GEO. For significant correlations with <span class="html-italic">p</span> &lt; 0.01, entire regression lines are shown, whereas for significant correlations with <span class="html-italic">p</span> &lt; 0.05, dashed regression lines are shown.</p>
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21 pages, 6915 KiB  
Article
NO Addition during Gas Oxygenation Reduces Liver and Kidney Injury during Prolonged Cardiopulmonary Bypass
by Aleksey Maksimovich Radovskiy, Andrey Evgenevich Bautin, Alexander Olegovich Marichev, Victor Vasilyevich Osovskikh, Natalia Yuryevna Semenova, Zoya Evgenyevna Artyukhina, Lada Aleksandrovna Murashova and Vsevolod Alexandrovich Zinserling
Pathophysiology 2023, 30(4), 484-504; https://doi.org/10.3390/pathophysiology30040037 - 19 Oct 2023
Viewed by 1284
Abstract
Objective. To evaluate the effect of NO added to the sweep gas of the oxygenator during cardiopulmonary bypass (CPB) on the liver and kidneys in pigs. Methods. An experiment was carried out on 10 pigs undergoing cardiac surgery using CPB. NO [...] Read more.
Objective. To evaluate the effect of NO added to the sweep gas of the oxygenator during cardiopulmonary bypass (CPB) on the liver and kidneys in pigs. Methods. An experiment was carried out on 10 pigs undergoing cardiac surgery using CPB. NO was added to the sweep gas of the oxygenator at a concentration of 100 ppm for the animals in the experimental group (CPB-NO, n = 5). Animals in the control group (CPB-contr, n = 5) did not receive NO in the sweep gas of the oxygenator. The CPB lasted 4 h, followed by postoperative monitoring for 12 h. To assess the injury to the liver and kidneys, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, creatinine, and neutrophil gelatinase-associated lipocalin (NGAL) were determined initially, at weaning from the CPB, and 6 and 12 h after weaning from the CPB. The glomerular filtration rate (GFR) was evaluated initially, at weaning from the CPB, and 6 and 12 h after weaning from the CPB. A pathomorphological study of the liver and kidneys was performed using semiquantitative morphometry. Results. The long four-hour period of CPB deliberately used in our experiment caused liver and kidney injury. In the CPB-contr group, an increase in the ALT concentration was found: 43 (34; 44) U/L at baseline to 82 (53; 99) U/L 12 h after CPB, p < 0.05. The AST concentration in the CPB-contr group increased from 25 (17; 26) U/L at baseline to 269 (164; 376) U/L 12 h after CPB, p < 0.05. We found no significant increase in the ALT and AST concentrations in the CPB-NO group. There were no significant differences in ALT and AST concentrations between the CPB-NO and CPB-contr groups at all the study time-points. In the CPB-contr group, an increase in the creatinine level was found from 131 (129; 133) µmol/L at baseline to 273 (241; 306) µmol/L 12 h after CPB, p < 0.05. We found no significant increase in creatinine level in the CPB-NO group. Creatinine levels in the CPB-NO group were significantly lower than in the CPB-contr group 12 h after weaning from CPB: 183 (168; 196) vs. 273 (241; 306) µmol/L; p = 0.008. The GFR in the CPB-NO group was significantly higher than in the CPB-contr group 6 h after weaning from CPB: 78.9 (77.8; 82.3) vs. 67.9 (62.3; 69.2) mL/min; p = 0.016. GFR was significantly higher in the CPB-NO group than in the CPB-contr group 12 h after weaning from CPB: 67.7 (65.5; 68.0) vs. 50.3 (48.7; 54.9) mL/min; p = 0.032. We found no significant differences between the study groups in the level of NGAL. We found several differences between the groups in the pathomorphological study. Conclusions. NO added to the sweep gas of the oxygenator reduces creatinine levels and increases GFR during prolonged CPB injury. Further research is required. Full article
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<p>Scheme of supply of air–oxygen mixture, NO, and monitoring of NO and NO<sub>2</sub> to the oxygenator.</p>
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<p>ALT dynamics in animals of the studied groups. The data are presented as a median (Q1; Q3); *—<span class="html-italic">p</span> &lt; 0.05 in comparison with the initial values.</p>
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<p>Dynamics of AST in animals of the studied groups. The data are presented as a median (Q1; Q3); *—<span class="html-italic">p</span> &lt; 0.05 in comparison with the initial values.</p>
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<p>Aggregation of erythrocytes in an animal from the CPB-contr group. Staining with H-E, 400×.</p>
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<p>Cells with bright-eosinophilic cytoplasm in the portal tract of an animal from the CPB-contr group. Staining with H-E, 400×.</p>
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<p>A vessel in the area of the expanded portal tract with moderate mononuclear infiltration in an animal of the CPB-contr group. Staining with H-E, 400×.</p>
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<p>Diffuse infiltration by neutrophils and lymphocytes in an animal of the CPB-contr group. Staining with H-E, 200×.</p>
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<p>Binuclear hepatocytes in an animal of the CPB-NO group. Staining with H-E, 200×.</p>
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<p>Dynamics of creatinine in animals of the studied groups. The data are presented as a median (Q1; Q3). *—<span class="html-italic">p</span> &lt; 0.05 in comparison with the initial values; #—<span class="html-italic">p</span> &lt; 0.01 in an intergroup comparison.</p>
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<p>Dynamics of NGAL in animals of the studied groups. The data are presented as a median (Q1; Q3). *—<span class="html-italic">p</span> &lt; 0.05 in comparison with the initial values.</p>
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<p>Alterative changes in the tubule epithelium in the renal cortex of a pig from the CPB-contr group (2 points, moderate changes). Staining with H-E, 400×.</p>
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<p>Alterative changes in the tubule epithelium in the renal cortex of the pig from the CPB-NO group (1 point, mild changes). Staining with H-E, 400×.</p>
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<p>Alterative changes in the vessel endothelium (2 points, moderate changes). Staining with H-E, 400×.</p>
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<p>Focal predominantly lymphocytic infiltration of the renal cortex in an animal of the CPB-contr group. Staining with H-E, 100×.</p>
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<p>Hyperemia of capillaries in renal medulla in an animal of the group, CPB-contr, erythrocytes forming “coin columns” (3 points, severe changes). Staining with H-E, 400×.</p>
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<p>Hyperemia of capillaries in renal medulla in an animal of the CPB-NO group (2 points, moderate changes) Staining with H-E, 400×.</p>
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13 pages, 2257 KiB  
Article
Essential Oils Produce Developmental Toxicity in Zebrafish Embryos and Cause Behavior Changes in Zebrafish Larvae
by Ivanildo Inacio da Silva, Niely Priscila Correia da Silva, James A. Marrs and Pabyton Gonçalves Cadena
Biomedicines 2023, 11(10), 2821; https://doi.org/10.3390/biomedicines11102821 - 18 Oct 2023
Viewed by 2027
Abstract
Essential oils have gained significant popularity in various industries due to their biological properties, but their potential toxic effects on living organisms have been poorly investigated. This study aimed to evaluate the effects of lemongrass, thyme, and oregano essential oils on zebrafish embryos [...] Read more.
Essential oils have gained significant popularity in various industries due to their biological properties, but their potential toxic effects on living organisms have been poorly investigated. This study aimed to evaluate the effects of lemongrass, thyme, and oregano essential oils on zebrafish embryos and larvae as animal models. Embryos were exposed to different concentrations of essential oils, and various endpoints were assessed, including epiboly, mortality (LC50), morphometry, and behavioral changes. All three essential oils reduced epiboly, affecting embryonic development. LC50 values were calculated for lemongrass (3.7 µg/mL), thyme (14.4 µg/mL), and oregano (5.3 µg/mL) oils. Larvae exposed to these oils displayed morphological defects, including growth reduction, spinal deformation, pericardial edema, eye size reduction, and reduced swim-bladder inflation. Morphometric analysis confirmed reduced larval length at higher oil concentrations. Essential-oil exposure altered zebrafish larval swimming behavior, with lemongrass oil reducing dark-cycle activity and oregano oil increasing light-cycle activity, suggesting neurodevelopmental toxicity. These findings illustrate the adverse effects of these oils on zebrafish embryos and larvae and reveal essential-oil toxicity, indicating careful use should be considered, particularly during pregnancy. Full article
(This article belongs to the Special Issue Zebrafish Models for Development and Disease 4.0)
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<p>Schematic representation of tests performed for toxicity evaluation of plant essential oils in embryo and larvae zebrafish.</p>
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<p>Epiboly percentages at 8 hpf in zebrafish embryos exposed to (<b>A</b>) lemongrass, (<b>B</b>) thyme, and (<b>C</b>) oregano essential oils. Test F by one-way ANOVA: lemongrass EO (F(5, 77) = 48.83, <span class="html-italic">p</span> &lt; 0.05); thyme EO (F(5, 76) = 48.97, <span class="html-italic">p</span> &lt; 0.05); and oregano EO (F(5, 72) = 29.95, <span class="html-italic">p</span> &lt; 0.05). * Statistically different from the control group (one-way ANOVA test, <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Representative zebrafish embryo epiboly and typical teratogenic effects observed in zebrafish embryos exposed to lemongrass, thyme, and oregano essential oils. Legend: (<b>A</b>) Control group epiboly at 8 hpf. (<b>B</b>) Thyme 25 µg/mL group epiboly at 8 hpf (scale bar 500 μm). (<b>C</b>,<b>D</b>) Fish affected by thyme essential-oil (20 µg/mL) exposure. Arrows and abbreviations indicate the main teratogenic effects observed as spine deformation (SD), small eyes (EY), pericardial edema (PE), swim-bladder inflation (SB), and growth retardation (GR). (<b>E</b>,<b>F</b>) Control group in lateral and dorsal views, respectively (scale bar 1 mm).</p>
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<p>Survival curve and the mortality linear regression of zebrafish embryos after exposure to lemongrass (LGEO), thyme (TEO), and oregano (OREO) essential oils (<b>A</b>, <b>B</b>, and <b>C</b>, respectively). The linear regression and relationship of probit essential-oil concentrations were used to calculate LC50 values in larvae at 6 days post-fertilization.</p>
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<p>Zebrafish larvae behavior in excitatory dark–light locomotor test. (<b>A</b>–<b>F</b>) Bars show the average of small (between 4 and 8 mm/s) or large (&gt;8 mm/s) activity movement for each dark or light period (10 min). (<b>G</b>–<b>L</b>) Average swimming behavior (2 min) in each complete dark–light cycle (20 min). Behavior test was analyzed by one-way ANOVA in light period (lemongrass EO small activity (F(2, 15) = 2.273, <span class="html-italic">p</span> = 0.14), large activity (F(2, 15) = 3.943, <span class="html-italic">p</span> &lt; 0.05); thyme EO small activity (F(2, 15) = 3.463, <span class="html-italic">p</span> = 0.06), large activity (F(2, 15) = 0.5043, <span class="html-italic">p</span> = 0.61); oregano EO small activity (F(2, 15) = 1.984, <span class="html-italic">p</span> = 0.17), large activity (F(2, 15) = 4.964, <span class="html-italic">p</span> &lt; 0.05)) and dark period (lemongrass EO small activity (F(2, 15) = 13.10, <span class="html-italic">p</span> &lt; 0.05), large activity (F(2, 15) = 12.77, <span class="html-italic">p</span> &lt; 0.05); thyme EO small activity (F(2, 15) = 2.829, <span class="html-italic">p</span> = 0.09), large activity (F(2, 15) = 2.547, <span class="html-italic">p</span> = 0.11); oregano EO small activity (F(2, 15) = 2.925, <span class="html-italic">p</span> = 0.08), large activity (F(2, 15) = 8.370, <span class="html-italic">p</span> &lt; 0.05)). When the time variable was considered, two-way ANOVA test was realized in small activity: lemongrass EO small activity (F(2, 69) = 9.015, <span class="html-italic">p</span> &lt; 0.05) and large activity (F(2, 69) = 9.924, <span class="html-italic">p</span> &lt; 0.05); thyme EO small activity (F(2, 69) = 1.436, <span class="html-italic">p</span> = 0.24) and large activity (F(2, 69) = 0.456, <span class="html-italic">p</span> = 0.64); oregano EO small activity (F(2, 69) = 1.951, <span class="html-italic">p</span> = 0.15) and large activity (F(2, 69) = 2.746, <span class="html-italic">p</span> = 0.07). * Represents statistically significant (<span class="html-italic">p</span> &lt; 0.05) difference by control group. <sup>#</sup> Represents statistically significant (<span class="html-italic">p</span> &lt; 0.05) difference by another essential-oil group.</p>
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20 pages, 7649 KiB  
Article
Adipocytes in the Uterine Wall during Experimental Healing and in Cesarean Scars during Pregnancy
by Natalia Tikhonova, Andrey P. Milovanov, Valentina V. Aleksankina, Ilyas A. Kulikov, Tatiana V. Fokina, Andrey P. Aleksankin, Tamara N. Belousova, Ludmila M. Mikhaleva and Natalya V. Niziaeva
Int. J. Mol. Sci. 2023, 24(20), 15255; https://doi.org/10.3390/ijms242015255 - 17 Oct 2023
Viewed by 1762
Abstract
We have suggested that adipocytes in uterine scars may affect the development of the placenta accrete spectrum (PAS). In the experimental part, we explored adipocytes in the uterine wall by the twelfth sexual cycle after surgery. In the clinical part, we investigated adipocyte [...] Read more.
We have suggested that adipocytes in uterine scars may affect the development of the placenta accrete spectrum (PAS). In the experimental part, we explored adipocytes in the uterine wall by the twelfth sexual cycle after surgery. In the clinical part, we investigated adipocyte clusters in the cesarean scar of pregnant women with and without PAS. The uterine wall was evaluated in gross and histological sections using morphometry, histochemistry (hematoxylin and eosin stain, Mallory stain), and immunohistochemistry for FABP4 (adipocyte markers), CD68, CD163, CD206 (macrophages), CD 34 (endothelium), cytokeratin 8 (epithelium), aSMA (smooth muscle cells). The design included an experimental study on Sprague–Dawley rats (n = 18) after a full-thickness surgical incision on the seventh (n = 6), 30th (n = 6), and 60th day (n = 6). The clinical groups include pregnant women without uterine scars (n = 10), pregnant women with a uterine scar after previous cesarean sections (n = 10), and women with PAS (n = 11). Statistical processing was carried out using nonparametric methods. Comparisons were conducted using the Mann–Whitney U-test and Kruskal–Wallis test. Statistical significance was considered at p < 0.05. On the seventh day, the rat uterine horn was enveloped by adipose tissue, which contained crown-like structures with FABP4+, CD68+, CD206+, and CD163+ cells. FABP4+ cells in the uterine wall were absent by the 30th day. The number of CD206+ and CD163+ cells in the adipose tissue decreased by the 30th day. On the 60th day, the attachment of fat tissue was revealed in the form of single strands. The serous layer around the damaged area totally recovered on the 60th day. FABP4+ cells were not detected in the uterine wall samples from pregnant women without a previous cesarean section. Adipocytes were found in the scar during non-complicated pregnancy and with PAS. Reducing the number of CD68+ cells in adipocyte clusters, there were in myometrium with PAS. Increased CD206+ and CD163+ cells were revealed in uterine adipocyte clusters of the group. According to the experimental finding, adipocytes should be absent in the uterine wall by the 12th sexual cycle after a full-thickness surgical incision. The presence of adipocyte clusters in cesarean scar indicated the disturbance of cell interaction. Differences in the numbers of CD206 and CD163 cells in adipocyte clusters between groups with and without PAS may be indirect evidence that uterine adipocytes affect the development of PAS. Full article
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<p>Gross picture of rat uterine horns after a full-thickness surgical incision. (<b>A1</b>–<b>A5</b>) Conducting a surgical incision of the uterine horn; (<b>B1</b>–<b>B3</b>) Fat tissue enveloping of the operated horn (left) on the 7th day (<b>B1</b>), 30th day (<b>B2</b>), and 60th day (<b>B3</b>); (<b>B4</b>) Covering the damaged area with fat tissue at different times after surgery; (<b>C1</b>–<b>C3</b>) The difference in length of the operated horn (left) compared to the intact one on the 7th day (<b>C1</b>), 30th day (<b>C2</b>), and 60th day (<b>C3</b>); (<b>C4</b>) The ratio of the operated horn length to the intact horn length at different times after surgery.</p>
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<p>The healing area of the operated rat uterine horn. (<b>A</b>) ×50, Mallory staining; (<b>B</b>) ×100, immunohistochemical staining on FABP4; (<b>C</b>) ×100, immunohistochemical staining on α-SMA, Mayer’s hematoxylin staining; (<b>D</b>) ×100, CD34 immunohistochemical staining, Mayer’s hematoxylin staining; (<b>E</b>) ×100, CD68 immunohistochemical staining, Mayer’s hematoxylin staining; (<b>F</b>) ×200, CD163 immunohistochemical staining, Mayer’s hematoxylin staining; (<b>G</b>) ×200, CD206 immunohistochemical staining, Mayer’s hematoxylin staining; (<b>A1</b>,<b>B1</b>,<b>C1</b>,<b>D1</b>,<b>E1</b>,<b>F1</b>,<b>G1</b>) 7th day after surgery; (<b>A2</b>,<b>B2</b>,<b>C2</b>,<b>D2</b>,<b>E2</b>,<b>F2</b>,<b>G2</b>) 30th day; (<b>A3</b>,<b>A4</b>) 60th day; (<b>H1</b>) The crown-like structures in the area of attachment of adipose tissue to the uterine wall; (<b>H2</b>) CD68 cells in the adipose tissue attached to the uterine wall on the 7th, 30th, and 60th days after surgery; (<b>H3</b>) CD206 cells in the adipose tissue attached to the uterine wall on the 7th, 30th, and 60th day after surgery; (<b>H4</b>) CD163 cells in the adipose tissue attached to the uterine wall on the 7th, 30th, and 60th days after surgery; (<b>H5</b>) CD34 cells in the adipose tissue attached to the uterine wall on the 7th, 30th and 60th days after surgery; (*) <span class="html-italic">p</span> &lt; 0.05; (**) <span class="html-italic">p</span> &lt; 0.01; (<span style="color:#0500D6">●</span>) The blue dot presents value for individual sample on the 7th day after the operation; (<span style="color:red">●</span>) The red dot presents value for individual sample on the 30th day after the operation; (<span style="color:#01BB03">●</span>) The green dot presents value for individual sample on the 60th day after the operation. The blue, red, green horizontal lines present the medians.</p>
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<p>The uterine wall of women near the placenta. (<b>A</b>,<b>B</b>) Mallory staining; (<b>A1</b>,<b>B1</b>,<b>C1</b>,<b>D1</b>) Uterus without previous cesarean section and uterine scar; (<b>A2</b>,<b>B2</b>,<b>C2</b>,<b>D2</b>) Uterus with caesarian scar and non-complicated pregnancy; (<b>A3</b>,<b>B3</b>,<b>C3</b>,<b>D3</b>) Uterus with PAS; (<b>A1</b>,<b>A3</b>,<b>B3,D1</b>–<b>D3</b>) ×50; (<b>A2</b>,<b>B2, C2</b>,<b>C3</b>) ×100; (<b>C1</b>) ×200; (<b>C1</b>–<b>C3</b>) CD68 immunohistochemical staining, Mayer’s hematoxylin staining; (<b>D1</b>–<b>D3</b>) FABP4 immunohistochemical staining, Mayer’s hematoxylin staining; (<b>E1</b>) CD68 cells in comparison groups; (<b>E2</b>) Adipocyte clusters in comparison groups; norma—the control group of women without previous cesarean section and uterine scar; scar—the group of women with a cesarean scar and non-complicated pregnancy; PAS—a group of women with PAS; (**) <span class="html-italic">p</span> &lt; 0.01; (****) <span class="html-italic">p</span> &lt; 0.001; (ns) not significant; (<span style="color:#0500D6">●</span>) The blue dot presents value for individual sample of the control group of women without previous cesarean section and uterine scar; (<span style="color:red">●</span>) The red dot presents value for individual sample of the group of women with a cesarean scar and non-complicated pregnancy; (<span style="color:#01BB03">●</span>) The green dot presents value for individual sample of a group of women with PAS. The blue, red, green horizontal lines present the medians.</p>
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<p>Adipocyte clusters in the uterine wall of pregnant women with scars in the case of women with non-complicated pregnancy and with PAS. ×200. (<b>A</b>) CD68 immunohistochemical staining, Mayer’s hematoxylin staining; (<b>B</b>) CD206 immunohistochemical staining, Mayer’s hematoxylin staining; (<b>C</b>) CD163 immunohistochemical staining, Mayer’s hematoxylin staining; (<b>A1</b>,<b>B1</b>,<b>C1</b>) Group with scar and non-complicated pregnancy; (<b>A2</b>,<b>B2</b>,<b>C2</b>) Group with PAS; (**) <span class="html-italic">p</span> &lt; 0.01; (****) <span class="html-italic">p</span> &lt; 0.001; (<span style="color:#0500D6">●</span>) The blue dot presents value for individual sample of a group of women with a cesarean scar and non-complicated pregnancy; (<span style="color:red">●</span>) The red dot presents value for individual sample of a group of women with PAS. The blue and red horizontal lines present the medians.</p>
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<p>The uteroplacental region with abnormal invasion of placental villi (placenta increta), ×50; (<b>A</b>–<b>C</b>,<b>F</b>). Mallory stain; (<b>D</b>,<b>E</b>). Staining with hematoxylin and eosin.</p>
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<p>Vessels in the uterine wall with placental villi invasion. ×50. α-SMA immunohistochemical staining, Mayer’s hematoxylin staining; (<b>A</b>). The vessel in the scar zone with thickened muscle wall, disintegration, and dissociation of muscle fibers; (<b>B</b>). Continuation of the uterine wall of (<b>A</b>), vessels with thickened muscle wall, disintegration and dissociation of muscle fibers of the scar area, clusters of adipocytes in the perivascular space, and perimetrium zone.</p>
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<p>The uteroplacental region with PAS, ×50. FABP4 immunohistochemical staining, Mayer’s hematoxylin staining; (<b>A</b>) Villi invaded the myometrium in the scar area, а cluster of adipocytes; (<b>B</b>) Adipocyte clusters in the perivascular space; (<b>C</b>) Adipocyte clusters in the perivascular space (continuation of the uterine wall shown in (<b>B</b>)); black arrow indicates a single adipocyte.</p>
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<p>Adipocyte clusters arranged along the serosa in the uteroplacental region with placental villi invaded, ×50. (<b>A</b>)—Mallory staining; (<b>B</b>)—aSMA immunohistochemical staining, Mayer’s hematoxylin staining; (<b>C</b>,<b>D</b>)—Cytokeratin 8 immunohistochemical staining.</p>
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<p>Adipocyte clusters located near the vessels in the uterine scar zone with pathological invasion of placental villi. Immunohistochemical staining with Mayer’s hematoxylin, ×200. (<b>A</b>,<b>C</b>,<b>E</b>,<b>G</b>)—Venous and arterial vessels inside the adipocyte cluster; (<b>B</b>,<b>D</b>,<b>F</b>,<b>H</b>)—Adipocyte cluster near the vessels; (<b>A</b>,<b>B</b>)—Staining for CD34; (<b>C</b>,<b>D</b>)—Staining for CD68; (<b>E</b>,<b>F</b>)—Staining for CD163; (<b>G</b>,<b>H</b>)—Staining for CD206.</p>
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<p>Vascular collectors of the uteroplacental region in the area of placental villus invasion. (<b>A</b>). Mallory staining, ×50; (<b>B</b>,<b>C</b>). CD34 immunohistochemical staining, ×100 (<b>B</b>), ×200 (<b>C</b>); (<b>D</b>,<b>E</b>). FABP4 immunohistochemical staining, ×200 (<b>D</b>), ×400 (<b>E</b>).</p>
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15 pages, 51528 KiB  
Article
Dendrological Secrets of the Pazaislis Monastery in Central Lithuania: DNA Markers and Morphology Reveal Tilia × europaea L. Hybrids of an Impressive Age
by Girmantė Jurkšienė, Darius Danusevičius, Rūta Kembrytė-Ilčiukienė and Virgilijus Baliuckas
Plants 2023, 12(20), 3567; https://doi.org/10.3390/plants12203567 - 13 Oct 2023
Viewed by 1230
Abstract
We benefited from the availability of a species-specific DNA marker to describe the morphometry of T. cordata × platyphyllos hybrids of an impressive age (ca. 150 years) grown in the Pazaislis baroque monastery yard in Central Lithuania. In an earlier study on a [...] Read more.
We benefited from the availability of a species-specific DNA marker to describe the morphometry of T. cordata × platyphyllos hybrids of an impressive age (ca. 150 years) grown in the Pazaislis baroque monastery yard in Central Lithuania. In an earlier study on a country-wide set of 543 T. cordata individuals from natural forest populations in Lithuania, we detected a nuclear microsatellite locus Tc8 well-differentiating between T. cordata and T. platyphyllos. The Tc8 locus contained a 140 bp allele in T. cordata (541 sampled individuals) and alleles above 160 bp in the two trees with a T. platyphyllos-like morphology (sampled in a national park). To verify the Tc8 locus as species specific, we sampled a further four T. platyphyllos-like individuals, which all contained the Tc8 locus alleles above 160 bp. We subsequently genotyped the six old-growth individuals from the Pazaislis monastery with mixed T. cordata × platyphyllos morphology. Results revealed that all six old-growth Tilia individuals from the Pazaislis monastery were heterozygous for the Tc8 locus with alleles of 140 bp (indicative of T. cordata) and 162 bp (indicative of T. platyphyllos). This finding confirms the morphological observations that these individuals are hybrids between T. cordata and T. platyphyllos. Additionally, the genotyping of a set of 14 nuclear microsatellite loci revealed that all six trees from the Pazaislis monastery are clones, possessing identical microsatellite genotypes. After the molecular identification, we morphotyped leaves, bracts, twigs, and nuts of the 6 old-growth T. cordata × platyphyllos hybrids from the Pazaislis monastery, 16 T. cordata old-growth trees, 4 T. × europaea var. europaea ‘Pallida’ trees growing near the Pazaislis monastery, and 4 mature T. platyphyllos trees from a nearby Girionys park. The morphotyping showed that T. cordata × platyphyllos hybrids may be the easiest to distinguish from T. cordata by raised and horizontally tertiary veins of leaves. Full article
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Graphical abstract

Graphical abstract
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<p>Location of the <span class="html-italic">Tilia</span> sp. individuals sampled in the two parks in central Lithuania: (<b>a</b>) location within Lithuania, (<b>b</b>) location in the city of Kaunas, (<b>c</b>) the samples in the Pazaislis park, (<b>d</b>) the samples in Girionys park. Species coded by cycle color (morphologically assigned): grey—<span class="html-italic">Tilia cordata</span> Mill.; green—<span class="html-italic">T. platyphyllos</span> Scop.; red—<span class="html-italic">T</span>. × <span class="html-italic">europaea</span> L.; yellow—<span class="html-italic">T</span>. × <span class="html-italic">europaea</span> var. <span class="html-italic">europaea</span> ‘Pallida’.</p>
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<p>Results of the principal component analysis (PCA) on the morphometric traits of <span class="html-italic">Tilia</span> leaves: (<b>a</b>) scatter plot of individual observations on <span class="html-italic">Tilia</span> specimens based on the two first principal components, (<b>b</b>) description of the principal components (Wicklin 2019). The abbreviations of <span class="html-italic">Tilia</span> species (S) are explained in <a href="#plants-12-03567-t001" class="html-table">Table 1</a>, and the abbreviations of the traits are in <a href="#plants-12-03567-t004" class="html-table">Table 4</a>.</p>
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<p>The morphometric traits of bracts (<b>a</b>) and leaves (<b>b</b>) of <span class="html-italic">Tilia</span> sp. measured with WinFolia (2016).</p>
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14 pages, 1550 KiB  
Article
The Use of Additives to Prevent Urolithiasis in Lambs Fed Diets with a High Proportion of Concentrate
by Vicente Luiz Macêdo Buarque, Helena Viel Alves Bezerra, Guilherme Pegoraro Rissi, Regner Ítalo Gonçalves de Oliveira, Natália Marques da Silva, Nara Regina Brandão Cônsolo, Germán Darío Ramírez-Zamudio, Ricardo de Francisco Strefezzi, Sarita Bonagurio Gallo, Saulo Luz Silva and Paulo Roberto Leme
Vet. Sci. 2023, 10(10), 617; https://doi.org/10.3390/vetsci10100617 - 11 Oct 2023
Viewed by 1805
Abstract
This study aimed to evaluate the effectiveness of different additives in preventing urolithiasis in lambs fed a diet rich in concentrate and their impact on performance and blood and urinary parameters. Thirty-two noncastrated male lambs, crossbred Dorper × Santa Inês, with initial body [...] Read more.
This study aimed to evaluate the effectiveness of different additives in preventing urolithiasis in lambs fed a diet rich in concentrate and their impact on performance and blood and urinary parameters. Thirty-two noncastrated male lambs, crossbred Dorper × Santa Inês, with initial body weights (BWs) of 23 ± 0.1 kg and ages of 50 ± 5 days, were kept in individual pens and fed a diet composed of 6% Cynodon ssp. hay and 94% concentrate and subjected to four treatments: CON without inclusion of additives, addition of ACL 5 g/kg of dry matter (DM), addition of CCL 6.3 g/kg of DM, and addition of BZA 5 g/kg of DM. There was no effect of treatment or interaction with time on blood parameters (p > 0.050), and performance characteristics, morphometry of ruminal papillae, and scores of cecum and rumen lesions were not affected by the addition of additives to the diet (p > 0.050). Greater urinary acidification was observed in animals from CCL and ACL treatments (p = 0.033). Calcium chloride acidified the urinary pH and can be used instead of ammonium chloride at a concentration of 0.63% based on DM, when this is the objective. Full article
(This article belongs to the Special Issue Effects of Nutrition on Ruminants Production Performance and Health)
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<p>Urinary pH of lambs fed a high-concentrate diet with or without added additives and days on feed. CON: control; ACL: ammonium chloride; CCL: calcium chloride; BZA: benzoic acid. T: treatments; D: days on feed; T × D: treatments and days on feed interaction. Similar letters do not differ from each other by the least significant difference T-Fisher (LSD) test at the 5% probability level.</p>
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<p>Ureter with the presence of stones and intense luminal stenosis in lamb. HE, obj. 4×. HE: hematoxylin and eosin; obj.4×: 4× magnification objective microscopic lens.</p>
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<p>Rumenitis score of lambs fed a high-concentrate diet with or without added additives. CON: control; ACL: ammonium chloride; CCL: calcium chloride; BZA: benzoic acid. Similar letters do not differ from each other by the least significant difference T-Fisher (LSD) test at the 5% probability level.</p>
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<p>Cecal lesion score of lambs fed a high-concentrate diet with or without added additives. CON: control; ACL: ammonium chloride; CCL: calcium chloride; BZA: benzoic acid. Similar letters do not differ from each other by the least significant difference T-Fisher (LSD) test at the 5% probability level.</p>
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12 pages, 3153 KiB  
Article
The Complementary Effects of Dabigatran Etexilate and Exercise Training on the Development and Stability of the Atherosclerotic Lesions in Diabetic ApoE Knockout Mice
by Nikolaos PE Kadoglou, Marianna Stasinopoulou, Evangelia Gkougkoudi, Eirini Christodoulou, Nikolaos Kostomitsopoulos and Georgia Valsami
Pharmaceuticals 2023, 16(10), 1396; https://doi.org/10.3390/ph16101396 - 2 Oct 2023
Cited by 1 | Viewed by 978
Abstract
Aim: To determine the complementary effects of dabigatran etexilate (DE), exercise training (ET), and combination (DE + ET) on the development and stability of the atherosclerotic lesions in diabetic apoE knockout (apoE−/−) mice. Methods: In 48 male apoE−/− diabetic mice, [...] Read more.
Aim: To determine the complementary effects of dabigatran etexilate (DE), exercise training (ET), and combination (DE + ET) on the development and stability of the atherosclerotic lesions in diabetic apoE knockout (apoE−/−) mice. Methods: In 48 male apoE−/− diabetic mice, streptozotocin (STZ) was induced for 5 consecutive days. Mice received a high-fat diet (HFD) for 8 weeks and then were randomized into four groups (1. Control/CG, 2. DEG: HFD with DE, 3. ETG: ET on treadmill, 4. DE + ETG: combination DE and ET treatment). At the end of the eighth week, all mice were euthanatized and morphometry of the aortic lesions at the level of aortic valve was obtained. Collagen, elastin, MCP-1, TNF-a, matrix metalloproteinases (MMP-2,-3,-9), and TIMP-1 concentrations within plaques at the aortic valve were determined. Results: All active groups had significantly smaller aorta stenosis (DEG:7.9 ± 2.2%, ETG:17.3 ± 5.3%, DE + ETG:7.1 ± 2.7%) compared to CG (23.3 ± 5.5% p < 0.05), reduced the relative intra-plaque content of MCP-1, macrophages, MMP-3, and MMP-9, and considerably increased collagen, elastin, and TIMP-1 (p < 0.05). Group 4 showed the most pronounced results (p < 0.05). Both DEG and DE + ETG significantly reduced MMP-2 and TNF-a concentrations compared to ETG and CG (p < 0.010). Conclusion: DE and ET treatment of diabetic apoE−/− mice resulted in complementary amelioration of atherosclerotic lesions development and stability, mediated by the anti-inflammatory modulation of both DE and ET. Full article
(This article belongs to the Section Medicinal Chemistry)
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<p>All active groups (dabigatran etexilate, exercise training, combined treatment) significantly reduced plaque formation compared to controls in ApoE<sup>−/−</sup> mice. Representative images and quantifications of aortic valve sections stained with hematoxylin/eosin, across all groups.</p>
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<p>All active groups (dabigatran etexilate, exercise training, combined treatment) significantly enhanced collagen concentrations compared to controls in ApoE<sup>−/−</sup> mice. Representative images of aortic valve sections stained with Sirius red (collagen) across all groups.</p>
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<p>Quantification of immunohistochemical staining with antibodies against MMP-2, MMP-3, MMP-9, TIMP-1, TNF-a, and MCP-1. * <span class="html-italic">p</span> &lt; 0.05 vs. CG, # <span class="html-italic">p</span> &lt; 0.05 vs. DEG, ‖ <span class="html-italic">p</span> &lt; 0.05 vs. ETG.</p>
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<p>Representative images of plaques at aortic valve sections stained with MMP-9 and MCP-1 across all groups. Section thickness was set at 5 μm and the original magnification was 100×. We analyzed 2–3 sections of each mouse (6 mice per group). CG, control group; DEG, dabigatran etexilate group; ETG, exercise training group; DE + ETG, dabigatran etexilate + exercise training group.</p>
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14 pages, 1758 KiB  
Article
The Effects of Prenatal Pravastatin Treatment in the Rabbit Fetal Growth Restriction Model
by Katerina Zapletalova, Ignacio Valenzuela, Marnel Greyling, Yannick Regin, Cristian Frigolett, Ladislav Krofta, Jan Deprest and Johannes van der Merwe
Biomedicines 2023, 11(10), 2685; https://doi.org/10.3390/biomedicines11102685 - 30 Sep 2023
Viewed by 1072
Abstract
Fetal growth restriction (FGR) remains without an effective prenatal treatment. Evidence from murine FGR models suggests a beneficial effect of prenatal pravastatin. Since the rabbit hemodichorial placenta more closely resembles the human condition, we investigated the effects of prenatal maternal pravastatin administration in [...] Read more.
Fetal growth restriction (FGR) remains without an effective prenatal treatment. Evidence from murine FGR models suggests a beneficial effect of prenatal pravastatin. Since the rabbit hemodichorial placenta more closely resembles the human condition, we investigated the effects of prenatal maternal pravastatin administration in the rabbit FGR model. At a gestational age of 25 days (term 31d), pregnant dams underwent partial uteroplacental vessel ligation (UPVL) in one uterine horn to induce FGR, leaving the other horn as a control. Dams were randomized to either receive 5 mg/kg/d pravastatin dissolved in their drinking water or normal drinking water until delivery. At GA 30d, the rabbits were delivered and were divided into four groups: control without pravastatin (C/NoPrav), FGR without pravastatin (FGR/NoPrav), FGR with pravastatin (FGR/Prav), and controls with pravastatin (C/Prav). The newborn rabbits underwent pulmonary functional assessment and neurobehavioral assessment, and they were harvested for alveolar morphometry or neuropathology. The placentas underwent histology examination and RNA expression. Birth weight was lower in the FGR groups (FGR/Prav, FGR/NoPrav), but there was no difference between FGR/Prav and C/NoPrav. No differences were noted in placental zone proportions, but eNOS in FGR/Prav placentas and VEGFR-2 in FGR/Prav and C/Prav were upregulated. There were no differences in pulmonary function assessment and alveolar morphometry. FGR/Prav kittens had increased neurosensory scores, but there were no differences in neuromotor tests, neuron density, apoptosis, and astrogliosis. In conclusion, in the rabbit FGR model, pravastatin upregulated the expression of VEGFR-2 and eNOS in FGR placentas and was associated with higher neurosensory scores, without measurable effects on birthweight, pulmonary function and morphology, and neuron density. Full article
(This article belongs to the Section Molecular and Translational Medicine)
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<p>Experimental setup.</p>
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<p>Birth weight comparison of newborn rabbits. C/NoPrav: Controls without pravastatin (33 subjects from 7 litters); FGR/NoPrav: FGR without pravastatin (36 subjects from 7 litters); FGR/Prav: FGR with pravastatin (30 subjects from 7 litters); C/Prav: controls with pravastatin (32 subjects from 7 litters). Data were compared using a linear mixed-model; graph shows mean ± SD; ns (not statistically significant<span class="html-italic">, p</span>-value &gt; 0.05).</p>
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<p>(<b>A</b>) Placental zone distribution by histopathological assessment. Data from 87 placentas from 14 litters: C/NoPrav: Controls without pravastatin (32 subjects from 7 litters); FGR/NoPrav: FGR without pravastatin (28 subjects from 7 litters); FGR/Prav: FGR with pravastatin (16 subjects from 7 litters); C/Prav: controls with pravastatin (21 subjects from 7 litters). Data were compared using mixed-effects analysis and are presented as mean ± SD. Only <span class="html-italic">p</span>-values &lt; 0.05 are shown. (<b>B</b>) Placentas of FGR/NoPrav and FGR/Prav kittens with cytokeratin/lectin double staining, divided by placental zones: decidua (D), junction zone (J), labyrinth (L).</p>
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<p>RNA expression of 51 placentas from 7 litters. C/NoPrav: Controls without pravastatin (8 subjects from 4 litters); FGR/NoPrav: FGR without pravastatin (20 subjects from 4 litters); FGR/Prav: FGR with pravastatin (10 subjects from 3 litters); C/Prav: controls with pravastatin (13 subjects from 3 litters). (<b>A</b>) VEGF-A: vascular endothelial growth factor; (<b>B</b>) VEGFR-2: vascular endothelial growth factor receptor 2; (<b>C</b>) eNOS: endothelial nitric oxide synthase; (<b>D</b>) SOD: superoxide dismutase;. Data analyzed using one-way ANOVA, shown as mean ± SD. Only <span class="html-italic">p</span>-values &lt; 0.05 are shown.</p>
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<p>Alveolar morphometry: (<b>A</b>) Alveolar size (Lm), (<b>B</b>) Alveolar airspace, (<b>C</b>) Alveolar wall thickness (Lmw) from 63 newborn rabbits from 8 litters. Data comparison was done using a mixed-effects model, with correction for multiple comparison using Tukey test. Data are presented as mean ± SD. C/NoPrav: Controls without pravastatin (17 subjects from 4 litters); FGR/NoPrav: FGR without pravastatin (16 subjects from 4 litters); FGR/Prav: FGR with pravastatin (17 subjects from 3 litters); C/Prav: controls with pravastatin (14 subjects from 3 litters).</p>
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<p>Neurodevelopmental assessment at PND 1 in postnatal day 1 rabbits. (<b>A</b>) Neurobehavioral tests grouped scores. (<b>B</b>) Cell density assessed in cresyl violet stained slides. Data were analyzed using a linear mixed-effects model and are displayed mean ± SD.</p>
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19 pages, 3840 KiB  
Article
Disrupted Gray Matter Networks Associated with Cognitive Dysfunction in Cerebral Small Vessel Disease
by Yian Gao, Shengpei Wang, Haotian Xin, Mengmeng Feng, Qihao Zhang, Chaofan Sui, Lingfei Guo, Changhu Liang and Hongwei Wen
Brain Sci. 2023, 13(10), 1359; https://doi.org/10.3390/brainsci13101359 - 22 Sep 2023
Viewed by 1280
Abstract
This study aims to investigate the disrupted topological organization of gray matter (GM) structural networks in cerebral small vessel disease (CSVD) patients with cerebral microbleeds (CMBs). Subject-wise structural networks were constructed from GM volumetric features of 49 CSVD patients with CMBs (CSVD-c), 121 [...] Read more.
This study aims to investigate the disrupted topological organization of gray matter (GM) structural networks in cerebral small vessel disease (CSVD) patients with cerebral microbleeds (CMBs). Subject-wise structural networks were constructed from GM volumetric features of 49 CSVD patients with CMBs (CSVD-c), 121 CSVD patients without CMBs (CSVD-n), and 74 healthy controls. The study used graph theory to analyze the global and regional properties of the network and their correlation with cognitive performance. We found that both the control and CSVD groups exhibited efficient small-world organization in GM networks. However, compared to controls, CSVD-c and CSVD-n patients exhibited increased global and local efficiency (Eglob/Eloc) and decreased shortest path lengths (Lp), indicating increased global integration and local specialization in structural networks. Although there was no significant global topology change, partially reorganized hub distributions were found between CSVD-c and CSVD-n patients. Importantly, regional topology in nonhub regions was significantly altered between CSVD-c and CSVD-n patients, including the bilateral anterior cingulate gyrus, left superior parietal gyrus, dorsolateral superior frontal gyrus, and right MTG, which are involved in the default mode network (DMN) and sensorimotor functional modules. Intriguingly, the global metrics (Eglob, Eloc, and Lp) were significantly correlated with MoCA, AVLT, and SCWT scores in the control group but not in the CSVD-c and CSVD-n groups. In contrast, the global metrics were significantly correlated with the SDMT score in the CSVD-s and CSVD-n groups but not in the control group. Patients with CSVD show a disrupted balance between local specialization and global integration in their GM structural networks. The altered regional topology between CSVD-c and CSVD-n patients may be due to different etiological contributions, which may offer a novel understanding of the neurobiological processes involved in CSVD with CMBs. Full article
(This article belongs to the Section Neurotechnology and Neuroimaging)
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<p>Flowchart for the construction of subject-wise GM morphological networks using T1-weighted MRI. (1) GM volume maps were created by segmenting, normalizing, modulating, and smoothing individual structural pictures using the VBM-DARTEL method. (2) GM volume map partitioned into 90 regions based on the AAL atlas. (3) Voxel-wise GMV for each region, retrieved and utilized to generate a histogram, and (4) the determined histogram distance (histogram intersection metric [<a href="#B26-brainsci-13-01359" class="html-bibr">26</a>]) between each pair of areas, resulting in a 90 × 90 similarity matrix. (5) An interval of 2% and a sparsity range from 10% to 60% used to threshold each matrix. Sparse network visualization is shown in the lateral perspective.</p>
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<p>Group comparisons of global topological properties among the three groups. Data points indicated with a star indicate the global network metric demonstrating significant differences between the CSVD-c/CSVD-n group and the control group under a corresponding sparsity threshold (<span class="html-italic">p</span> &lt; 0.05, ANCOVA with LSD post hoc test). The CSVD-c and CSVD-n groups showed no differences.</p>
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<p>Hub region distributions of the GM networks for both groups. The hub nodes’ various node sizes are displayed alongside their nodal betweenness centrality scores. Software called BrainNet Viewer V1.6 (<a href="http://www.nitrc.org/projects/bnv/" target="_blank">http://www.nitrc.org/projects/bnv/</a>, accessed on 12 May 2021) was used to display the brain graphs. Please see <a href="#app1-brainsci-13-01359" class="html-app">Supplementary Table S1</a> for a list of node acronyms.</p>
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<p>The differences in nodal betweenness centrality of the GM networks among the three groups. The scaled node sizes represent the F values in the ANOVA, and the disrupted nodes with substantially altered nodal betweenness centrality are represented in blue or red, respectively. Please see <a href="#app1-brainsci-13-01359" class="html-app">Supplementary Table S1</a> for a list of node acronyms.</p>
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<p>Scatter plots showing the significant Pearson’s correlations between network topological properties (Eglob, Eloc, and Lp) and SDMT scores for the (<b>A</b>) CSVD−c (red) and (<b>B</b>) CSVD−n (orange) groups. One subject is indicated with each dot. We give linear regression lines, r (partial correlation coefficient), <span class="html-italic">p</span> values (FDR adjusted), and 95% confidence intervals for the best−fit line (shading area). In contrast, the scatter plots of the control group (blue) are also shown in each subgraph.</p>
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<p>Pearson’s correlation coefficients between the network topological properties and cognitive parameters in both the CSVD and control groups. This heatmap displays the correlation coefficient between global/regional metrics and cognitive test scores for the CSVD-c (<b>A</b>), CSVD-n (<b>B</b>), and control (<b>C</b>) groups. *: <span class="html-italic">p</span> &lt; 0.05, **: <span class="html-italic">p</span> &lt; 0.01. Higher scores on the TMT (B-A) and SCWT were associated with worse symptoms. Conversely, higher scores on the MoCA, AVLT, and SDMT were associated with better symptoms. For both groups, global/local efficiency (E<sub>glob</sub>, E<sub>loc</sub>) was negatively correlated with MoCA, AVLT, and SDMT scores and positively correlated with SCWT and TMT (B-A) scores, while shortest path length (L<sub>p</sub>) was the reverse. For the abbreviations of nodes, please see <a href="#app1-brainsci-13-01359" class="html-app">Supplementary Table S1</a>.</p>
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14 pages, 9018 KiB  
Article
Resolving the Puzzle of Iris maackii (Iridaceae): A Morphological Insight into Its Taxonomy
by Eugeny V. Boltenkov
Plants 2023, 12(19), 3349; https://doi.org/10.3390/plants12193349 - 22 Sep 2023
Cited by 1 | Viewed by 1359
Abstract
Since the early 20th century, Iris maackii (Iridaceae) has been considered a synonym of I. laevigata, a synonym of I. pseudacorus, or an accepted species. The current concept of I. maackii in the literature and databases is often applied to yellow-flowered [...] Read more.
Since the early 20th century, Iris maackii (Iridaceae) has been considered a synonym of I. laevigata, a synonym of I. pseudacorus, or an accepted species. The current concept of I. maackii in the literature and databases is often applied to yellow-flowered plants with prominently veined rosette leaves, which are diagnostic features of I. pseudacorus growing in Northeast Asia. Therefore, the objective was to clarify the taxonomic identity of I. maackii. This study is based on a critical examination of the literature, on the observed morphological characters in the holotype of I. maackii, and on a morphological comparison of I. maackii with living plants of I. laevigata and I. pseudacorus. Additionally, a morphometric comparison of the seed characters was carried out to clarify the morphological distinction among I. maackii, I. laevigata, and I. pseudacorus. A careful study demonstrated that the rosette leaf texture and the morphology of the flowering stem, fruit, and seeds of I. maackii are identical to or within the variation range of I. laevigata. Thus, I. maackii is morphologically non-distinct from I. laevigata and should be recognized as a taxonomic synonym of the latter. An image of the holotype of I. maackii is provided along with detailed illustrations of I. laevigata and I. pseudacorus. Full article
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<p>Holotype of <span class="html-italic">Iris maackii</span> (LE01010783) (included with the permission of the curator).</p>
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<p>Images of the <span class="html-italic">Iris</span> species studied: a flower (<b>a</b>) and a habitat (<b>b</b>) of <span class="html-italic">I. laevigata</span> on a floating mat near Rudnev Bay, Primorsky Krai, Russia (42°55′10″ N 132°28′40″ E); a flower (<b>c</b>) and a habitat (<b>d</b>) of <span class="html-italic">I. pseudacorus</span> in the Tuzlov River, Rostov Oblast, Russia (47°28′15″ N 39°27′59″ E). Photos by the author.</p>
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<p>Morphological characters of <span class="html-italic">Iris laevigata</span> and <span class="html-italic">I. pseudacorus</span>: (<b>a</b>) flowering stems of <span class="html-italic">I. laevigata</span> (marks are as follows: 1, stem; 2, lateral shoot; 3, upper cauline leaf; 4, outer bract; 5, fruit; 6, pedicel; 7, terminal cluster; 8, lateral cluster); (<b>b</b>) flowering stems of <span class="html-italic">I. pseudacorus</span> (arrow indicates the inconspicuous lateral shoot); (<b>c</b>) a middle part of the rosette leaves (×, <span class="html-italic">I. laevigata</span>; ××, <span class="html-italic">I. pseudacorus</span>; arrows indicate the prominent midrib); (<b>d</b>) fruit (left row, <span class="html-italic">I. laevigata</span>; right row, <span class="html-italic">I. pseudacorus</span>); and (<b>e</b>) seeds (1–8 are collection site numbers; see <a href="#plants-12-03349-t001" class="html-table">Table 1</a>). Photos by the author. Images were taken using an ObjectScan 1600 scanner (Microtek International Inc., Taiwan).</p>
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<p>Whisker plots showing the results of Dunnett’s test of differences in the mean values of length (<b>a</b>), width (<b>b</b>), thickness (<b>c</b>), and L/W ratio (<b>d</b>) between the <span class="html-italic">Iris laevigata</span> and <span class="html-italic">I. pseudacorus</span> collection sites (see <a href="#plants-12-03349-t002" class="html-table">Table 2</a>) and <span class="html-italic">I. maackii</span>, selected as a control. The results of the test are presented as <span class="html-italic">p</span>-values, differences between the mean values of each experimental group (black dots), and 95% confidence intervals of these differences (whiskers) for each pairwise comparison. X-axis is differences in means. Dash-dotted line indicates zero difference. See <a href="#app1-plants-12-03349" class="html-app">Table S3</a> for more details.</p>
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<p>Principal component analysis of the morphometric parameters of seeds from <span class="html-italic">Iris maackii</span> (red), <span class="html-italic">I. laevigata</span> (blue), and <span class="html-italic">I. pseudacorus</span> (orange). Ellipses show 95% high-density regions for normal distributions representing two groups. Arrows indicate contribution of each morphometric parameter. The codes of the morphological characters of seeds are as follows: L, length; W, width; T, thickness; and L/W, length-to-width ratio. See <a href="#app1-plants-12-03349" class="html-app">Table S4</a> for more details.</p>
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