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Search Results (1,146)

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24 pages, 8840 KiB  
Article
Durability Requirements for Reinforced Concrete Structures Placed in a Hostile Tropical Coastal Environment
by Abel Castañeda Valdés, Francisco Corvo Pérez, Ildefonso Pech Pech, Rigoberto Marrero Águila and Emilio Bastidas-Arteaga
Buildings 2024, 14(8), 2494; https://doi.org/10.3390/buildings14082494 - 12 Aug 2024
Viewed by 214
Abstract
In this work, a series of durability requirements are proposed for the construction of long-service-life reinforced concrete (RC) structures in a coastal environment with extreme atmospheric corrosivity. RC specimens were exposed in a coastal outdoor site in Cuba for three years. Carbon steel [...] Read more.
In this work, a series of durability requirements are proposed for the construction of long-service-life reinforced concrete (RC) structures in a coastal environment with extreme atmospheric corrosivity. RC specimens were exposed in a coastal outdoor site in Cuba for three years. Carbon steel corrosion evaluation revealed an annual average atmospheric corrosion rate over the maximum limit established (ISO 9223:2012) for extreme (CX) atmospheric corrosivity. The service life of the RC structures, considered as the sum of the time-to-corrosion-initiation and time-to-corrosion-induced concrete cracking, was determined as a function of durability requirements. The most important durability requirements to achieve a long service life (>70 years) in RC structures subjected to a CX corrosivity category were defined as follows: water/cement ratio, compressive strength, percentage of effective capillary porosity, and concrete cover thickness. Under these hostile environments, the expansion of the corrosion products formed on the reinforcement steel and the induced cracking of the concrete could be attributed partially to the formation of the akaganeite phase in reinforcement steel, which revealed a different morphology compared to the akaganeite typically formed in bare carbon steels. Full article
(This article belongs to the Collection Advanced Concrete Materials in Construction)
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Figure 1
<p>Location of the COES in Havana, Cuba (<b>a</b>,<b>b</b>). MC means Meteorological Center in <b>b</b>. Dry plates and cellulose filters placed on a wooden rack (<b>c</b>). RC specimens (<b>d</b>). <span class="html-italic">Ic</span> measurement device (<b>e</b>).</p>
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<p>Geometry of RC specimens considered in this study.</p>
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<p>Behavior of monthly mean values of <span class="html-italic">Cl<sup>−</sup>DR</span> and <span class="html-italic">SOx<sup>−</sup>DR</span> between the cold and summer seasons during the first year of study.</p>
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<p>Monthly behavior of <span class="html-italic">RH</span>, <span class="html-italic">T</span>, and <span class="html-italic">WS</span> during the first year of study.</p>
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<p>Behavior of annual average values of the twelve <span class="html-italic">Ic</span> values versus time of exposure (R = reference, 1 = first year, 2 = second year, and 3 = third year). <span class="html-italic">CCTh</span> = 20 mm (<b>a</b>). <span class="html-italic">CCTh</span> = 40 mm (<b>b</b>).</p>
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<p>Prediction of <span class="html-italic">Ic</span> for concrete with <span class="html-italic">w/c</span> ratio of 0.4 versus exposure time according to the fitted model.</p>
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<p>Behavior of the twelve <span class="html-italic">Ic</span> values determined in the third year of exposure in the RC specimens as a function of the <span class="html-italic">w/c</span> ratios, <span class="html-italic">CCTh</span>, and the twelve values of <span class="html-italic">f<sub>ck</sub></span>, <span class="html-italic">ε<sub>e</sub></span>, <span class="html-italic">UPS</span>, and <span class="html-italic">D.</span> Changes in <span class="html-italic">Ic</span> vs. compressive strength (<b>a</b>), percentage of effective capillary porosity (<b>b</b>), ultrasonic pulse velocity (<b>c</b>), and density (<b>d</b>) for different <span class="html-italic">w/c</span> ratios and <span class="html-italic">CCTh</span>.</p>
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<p>Visual observation of RC specimens placed at COES during the three years of study.</p>
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<p>Visual observation of two specimens of carbon steel. Intense deterioration due to atmospheric corrosion is observed. The metal has been perforated by atmospheric corrosion.</p>
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<p>Different crystalline phases identified in the rust layers of reinforcement steel embedded in the RC specimen with a <span class="html-italic">w/c</span> ratio of 0.6 and a <span class="html-italic">CCTh</span> value of 20 mm placed at the COES until the third year of exposure.</p>
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<p>Morphology of the crystalline phases identified in the oxide layer formed on reinforcement steel surface. Lepidocrocite in foam form, goethite in flower form, and magnetite in ring form (<b>b</b>). Akaganeite in reinforcement bars formed with right-angled edges (<b>a</b>,<b>c</b>,<b>d</b>), EDS (<b>e</b>,<b>f</b>).</p>
Full article ">
14 pages, 3504 KiB  
Communication
Connexin 43 Modulation in Human Chondrocytes, Osteoblasts and Cartilage Explants: Implications for Inflammatory Joint Disorders
by Elena Della Morte, Chiara Giannasi, Alice Valenza, Francesca Cadelano, Alessandro Aldegheri, Luigi Zagra, Stefania Niada and Anna Teresa Brini
Int. J. Mol. Sci. 2024, 25(15), 8547; https://doi.org/10.3390/ijms25158547 - 5 Aug 2024
Viewed by 372
Abstract
Connexin 43 (Cx43) is crucial for the development and homeostasis of the musculoskeletal system, where it plays multifaceted roles, including intercellular communication, transcriptional regulation and influencing osteogenesis and chondrogenesis. Here, we investigated Cx43 modulation mediated by inflammatory stimuli involved in osteoarthritis, i.e., 10 [...] Read more.
Connexin 43 (Cx43) is crucial for the development and homeostasis of the musculoskeletal system, where it plays multifaceted roles, including intercellular communication, transcriptional regulation and influencing osteogenesis and chondrogenesis. Here, we investigated Cx43 modulation mediated by inflammatory stimuli involved in osteoarthritis, i.e., 10 ng/mL Tumor Necrosis Factor alpha (TNFα) and/or 1 ng/mL Interleukin-1 beta (IL-1β), in primary chondrocytes (CH) and osteoblasts (OB). Additionally, we explored the impact of synovial fluids from osteoarthritis patients in CH and cartilage explants, providing a more physio-pathological context. The effect of TNFα on Cx43 expression in cartilage explants was also assessed. TNFα downregulated Cx43 levels both in CH and OB (−73% and −32%, respectively), while IL-1β showed inconclusive effects. The reduction in Cx43 levels was associated with a significant downregulation of the coding gene GJA1 expression in OB only (−65%). The engagement of proteasome in TNFα-induced effects, already known in CH, was also observed in OB. TNFα treatment significantly decreased Cx43 expression also in cartilage explants. Of note, Cx43 expression was halved by synovial fluid in both CH and cartilage explants. This study unveils the regulation of Cx43 in diverse musculoskeletal cell types under various stimuli and in different contexts, providing insights into its modulation in inflammatory joint disorders. Full article
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Figure 1
<p>(<b>A</b>,<b>B</b>). Expression of Cx43 in TNFα and/or IL-1β-stimulated human articular chondrocytes (CHs) (<b>A</b>) and human osteoblasts (OBs) (<b>B</b>) at day 3, analyzed using Western blot. Specific bands were quantified through Image Lab Software v 6.1 (Bio-Rad, Milan, Italy) and data (<span class="html-italic">n</span> = 5 independent experiments/donors, each indicated by distinct dots) were normalized on ACTB and expressed as relative values (CTR = 1). The panels below show representative immunoblots. Statistical analysis was performed via one-way analysis of variance (ANOVA) using Tukey’s post hoc test. Data are shown as mean ± SD. Significances vs. CTR are shown as * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001; vs. IL # <span class="html-italic">p</span> ≤ 0.05.</p>
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<p>(<b>A</b>–<b>C</b>). Gene expression of GJA1 (<b>A</b>), MMP3 (<b>B</b>) and MMP13 (<b>C</b>) in TNFα-stimulated OB at day 1 analyzed using real-time PCR. Data (<span class="html-italic">n</span> = 6 independent experiments/donors, each indicated by distinct dots) are expressed as 2<sup>−ΔΔCt</sup> (TBP was used as a housekeeping gene). (<b>D</b>). Expression of Cx43 in OB pre-treated for 1 h with MG-132 and then stimulated with TNFα for 3 days, analyzed using Western blot. Specific bands were quantified through Image Lab Software v 6.1 (Bio-Rad, Milan, Italy) and data (<span class="html-italic">n</span> = 4 independent experiments/donors) were normalized on ACTB and expressed as relative values (CTR = 1). Statistical analyses were performed using paired <span class="html-italic">t</span>-test (<b>A</b>–<b>C</b>) or one-way analysis of variance (ANOVA) using Tukey’s post hoc test (<b>D</b>). Data are shown as mean ± SD. Significance vs. CTR is shown as * <span class="html-italic">p</span> ≤ 0.05 and **** <span class="html-italic">p</span> &lt; 0.0001.</p>
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<p>Laser scanning confocal microscopy of OB; Cx43 and β-Tubulin were revealed with an Alexa Fluor<sup>®</sup> 488 (Cx43) and 568 (β-tubulin) conjugated antibody (green and red respectively), while nuclei were stained with DAPI (blue) (magnification 63×). The scale bar indicates 10 µm and the orthogonal views (yellow dashed lines) were obtained using Fiji software (ImageJ 1.51).</p>
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<p>(<b>A</b>). Gene expression of GJA1 in CH treated with synovial fluid (SF50%) for 1 day analyzed using real-time PCR. Data (<span class="html-italic">n</span> = 4 independent experiments/donors, each indicated by distinct dots) are expressed as 2<sup>−ΔΔCt</sup> (TBP was used as a housekeeping gene). (<b>B</b>). Expression of Cx43 in CH treated with SF50% for 3 days analyzed using Western blot. A representative immunoblot is shown. Specific bands were quantified through Image Lab Software v 6.1 (Bio-Rad, Milan, Italy) and data (<span class="html-italic">n</span> = 4 independent experiments) were normalized on ACTB and expressed as relative values (CTR = 1). Data are shown as mean ± SD. Statistical analysis was performed using paired <span class="html-italic">t</span>-test. Significance vs. CTR is shown as * <span class="html-italic">p</span> ≤ 0.05 and *** <span class="html-italic">p</span> &lt; 0.001.</p>
Full article ">Figure 5
<p>(<b>A</b>,<b>B</b>). Expression of Cx43 in cartilage explants treated with TNFα (<b>A</b>) or SF50% (<b>B</b>) for 3 days analyzed using Western blot. Representative immunoblots are shown. Specific bands were quantified through Image Lab Software v 6.1 (Bio-Rad, Milan, Italy) and data (<span class="html-italic">n</span> = 8 and <span class="html-italic">n</span> = 5 independent experiments/donors, each indicated by distinct dots) were normalized on ACTB and expressed as relative values (CTR = 1). Data are shown as mean ± SD. Statistical analysis was performed using paired <span class="html-italic">t</span>-test. Significance vs. CTR is shown as * <span class="html-italic">p</span> ≤ 0.05.</p>
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15 pages, 6364 KiB  
Article
Microstructure and Wear Resistance of In Situ Synthesized Ti(C, N) Ceramic-Reinforced Nickel-Based Coatings by Laser Cladding
by Juncai Li, Ying Chen, Chuang Guan, Chao Zhang, Ji Zhao and Tianbiao Yu
Materials 2024, 17(15), 3878; https://doi.org/10.3390/ma17153878 - 5 Aug 2024
Viewed by 410
Abstract
In recent years, laser cladding technology has been widely used in surface modification of titanium alloys. To improve the wear resistance of titanium alloys, ceramic-reinforced nickel-based composite coatings were prepared on a TC4 alloy substrateusing coaxial powder feeding laser cladding technology. Ti (C, [...] Read more.
In recent years, laser cladding technology has been widely used in surface modification of titanium alloys. To improve the wear resistance of titanium alloys, ceramic-reinforced nickel-based composite coatings were prepared on a TC4 alloy substrateusing coaxial powder feeding laser cladding technology. Ti (C, N) ceramic was synthesized in situ by laser cladding by adding different contents (10%, 20%, 30%, and 40%) of TiN, pure Ti powder, graphite, and In625 powder. Thisestudy showed that small TiN particles were decomposed and directly formed the Ti (C, N) phase, while large TiN particles were not completely decomposed. The in situ synthetic TiCxN1−x phase was formed around the large TiN particles. With the increase in the proportion of powder addition, the wear volume of the coating shows a decreasing trend, and the wear resistance of the surface coating is improving. The friction coefficient of the sample with 40% TiN, pure Ti powder, and graphite powder is 0.829 times that of the substrate. The wear volume is 0.145 times that of the substrate. The reason for this is that with the increase in TiN, Ti, and graphite in the powder, there are more ceramic phases in the cladding layer, and the hard phases such as TiC, Ti(C, N) and Ti2Ni play the role in the structure of the “backbone”, inhibit the damage caused by micro-cutting, and impede the movement of the tearing point of incision, so that the coating has a higher abrasion resistance. Full article
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<p>Experimental setup diagram of laser cladding system.</p>
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<p>Preparation process of test samples.</p>
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<p>Temperature—dependent Gibbs free energy curves for various potential reactions during the solidification process.</p>
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<p>X-ray diffraction (XRD) patterns of different samples.</p>
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<p>EDS results of sample 1#: (<b>a</b>) electronic image, (<b>b</b>) EDS layered image, (<b>c</b>) Ti element, (<b>d</b>) N element, (<b>e</b>) C element, (<b>f</b>) Ni element, (<b>g</b>) Cr element, (<b>h</b>) Nb element.</p>
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<p>EDS surface scan image of sample 4#: (<b>a</b>) electronic image, (<b>b</b>) EDS layered image, (<b>c</b>) Ti element, (<b>d</b>) N element, (<b>e</b>) C element, (<b>f</b>) Ni element, (<b>g</b>) Cr element, (<b>h</b>) Nb element.</p>
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<p>Local line scanning results of sample 4.</p>
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<p>Friction coefficients of the matrix and various samples.</p>
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<p>Wear surface morphology of the substrate and various samples.</p>
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<p>Histograms of wear volume of the substrate and each sample.</p>
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<p>Microhardness and average microhardness on TC4 substrate and specimens 1#–4#.</p>
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<p>Optical surface morphology of abrasion marks on TC4 substrate and specimens 1#–4#.</p>
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14 pages, 2532 KiB  
Article
The Mechanical and Electrochemical Stability of Trimethysilane Plasma Nanocoatings Deposited onto Cobalt Chromium Cardiovascular Stents
by ThiThuHa Phan, John E. Jones, Yixuan Liao, Qingsong Yu and Meng Chen
Materials 2024, 17(15), 3699; https://doi.org/10.3390/ma17153699 - 26 Jul 2024
Viewed by 372
Abstract
The objective of this study was to evaluate the coating integrity performance and corrosion protection property of trimethylsilane (TMS) plasma nanocoatings that were directly deposited onto cobalt chromium (CoCr) L605 cardiovascular stents. Hydrophilic surfaces were achieved for the TMS plasma nanocoatings that were [...] Read more.
The objective of this study was to evaluate the coating integrity performance and corrosion protection property of trimethylsilane (TMS) plasma nanocoatings that were directly deposited onto cobalt chromium (CoCr) L605 cardiovascular stents. Hydrophilic surfaces were achieved for the TMS plasma nanocoatings that were deposited onto the coronary stents through NH3/O2 (2:1 molar ratio) plasma post-treatment. With a coating thickness of approximately 20–25 nm, the TMS plasma nanocoatings were highly durable and able to resist delamination and cracking from crimping and expansion by a Model CX with a J-Crimp Station. The stent surface that was evaluated by Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray Spectroscopy (EDS) showed no indications of pitting, corrosion, or adsorption products on either the luminal or abluminal surfaces of the stents, in contrast to the uncoated stent surface. The TMS plasma nanocoatings significantly enhanced the stent’s corrosion resistance in immersion experiments that followed the ASTM F2129-15 corrosion protocol, evident in the increase of the open circuit potential (OCP) from 0.01 V for the uncoated L605 stent to 0.18 V for the plasma-nanocoated L605 stent, reducing potential cytotoxic metal ion release. Cyclic polarization (CP) curves show that the corrosion rate (density level) observed in plasma-nanocoated L605 stents was approximately half an order of magnitude lower than that of the uncoated stents, indicating improved corrosion protection of the stents. CP curves of the TMS plasma-nanocoated stents with different coating thicknesses show that, in the range of 20–65 nm, the coating thickness does not result in any difference in the corrosion resistance of the stents. Full article
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<p>(<b>A</b>) Optical images of spherical water droplets on uncoated and plasma-nanocoated L605 stents. TMS+NH<sub>3</sub>/O<sub>2</sub> nanocoatings are shown after 1-day and 2-year aging periods. (<b>B</b>) Surface static contact angles for uncoated and TMS+NH<sub>3</sub>/O<sub>2</sub> plasma-nanocoated L605 coupons aged for up to 8 weeks, with six stents per group. S<sub>01</sub>, S<sub>10</sub>, and S<sub>21</sub> samples were designated for NH<sub>3</sub>/O<sub>2</sub> plasma post-treatment with NH<sub>3</sub>:O<sub>2</sub> gas ratios of 0:1, 1:0, and 2:1, respectively.</p>
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<p>SEM images of (<b>A</b>) coating failures following the dilatation process on plasma-nanocoated L605 stents at different coating thicknesses; (<b>B</b>) stent surfaces after tracking fixture test, in which the coating thickness was 20–25 nm for TMS+NH<sub>3</sub>/O<sub>2</sub> plasma nanocoatings.</p>
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<p>(<b>A</b>) SEM images for uncoated and TMS+NH<sub>3</sub>/O<sub>2</sub> plasma-nanocoated L605 stents after 7-day immersion test in flow conditions; (<b>B</b>) EDS spectra of adsorbed mineral products on the stents. The TMS+NH<sub>3</sub>/O<sub>2</sub> plasma nanocoating thickness was 20–25 nm.</p>
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<p>Electrochemical curves for uncoated and TMS+NH<sub>3</sub>/O<sub>2</sub> plasma-nanocoated L605 stents and coupons: (<b>A</b>) Open circuit potential with immersion time; (<b>B</b>) Cyclic polarization (CP) curves; (<b>C</b>) SEM images for surface morphology of uncoated and plasma-nanocoated L605 stents after CP test; (<b>D</b>) CP curves of plasma-nanocoated coupons with different coating thicknesses.</p>
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18 pages, 4579 KiB  
Article
Larval Competition between Aedes and Culex Mosquitoes Carries over to Higher Arboviral Infection during Their Adult Stage
by Adwine Vanslembrouck, Stephanie Jansen, Jacobus De Witte, Corneel Janssens, Stien Vereecken, Michelle Helms, Unchana Lange, Renke Lühken, Jonas Schmidt-Chanasit, Anna Heitmann and Ruth Müller
Viruses 2024, 16(8), 1202; https://doi.org/10.3390/v16081202 - 26 Jul 2024
Viewed by 843
Abstract
The common house mosquito (Culex pipiens) is a native vector for West Nile virus (WNV). Invasive species like the tiger mosquito (Aedes albopictus) and Asian bush mosquito (Aedes japonicus) are rapidly spreading through Europe, posing a major [...] Read more.
The common house mosquito (Culex pipiens) is a native vector for West Nile virus (WNV). Invasive species like the tiger mosquito (Aedes albopictus) and Asian bush mosquito (Aedes japonicus) are rapidly spreading through Europe, posing a major threat as vectors for dengue, chikungunya (CHIKV), and Japanese encephalitis virus (JEV). These mosquitoes share a similar ecological niche as larvae, but the carry-over effects of aquatic larval interactions to the terrestrial adult stage remain largely unknown and their medical relevance requires further investigation. This study examines the context dependency of larval interactions among Aedes albopictus, Aedes japonicus, and Culex pipiens. The survival, development time, growth, and energetic storage were measured in different European populations within density-response (intraspecific) experiments and replacement (interspecific) experiments at 20 °C and 26 °C. Overall, Ae. japonicus was the weakest competitor, while competition between Ae. albopictus and Cx. pipiens varied with temperature. Adults emerging from this larval competition were infected as follows: Culex pipiens with WNV, Ae. albopictus with CHIKV, and Ae. japonicus with JEV. While no JEV infection was observed, mosquitoes experiencing interspecific interactions during their larval stages exhibited higher infection rates and viral RNA titers for CHIKV and WNV. This increased susceptibility to viral infection after larval competition suggests a higher risk of arbovirus transmission in co-occurring populations. Full article
(This article belongs to the Section Insect Viruses)
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<p>Relative crowding coefficient with advantage or disadvantage. (<b>a</b>) Development time for 50% of the pupae to emerge, (<b>b</b>) the pupal size, (<b>c</b>) lipid, (<b>d</b>) glycogen, (<b>e</b>) protein content (size-corrected) of combination (1) <span class="html-italic">Ae. albopictus</span> vs. <span class="html-italic">Cx. pipiens</span> s.s./<span class="html-italic">Cx. torrentium</span> and (2) <span class="html-italic">Ae. japonicus</span> vs. <span class="html-italic">Cx. p. molestus</span> during interspecific competition at 20 °C and 26 °C. In red are the differences, with one of the species having a RCC below 1 indicating out-competition, according to Oberg, Young [<a href="#B51-viruses-16-01202" class="html-bibr">51</a>].</p>
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<p>Relative crowding coefficient and its effect on activity or inactivity. Behavior variables: total distance moved, velocity, and duration of body contact for intra- and interspecific larval competition ratios of <span class="html-italic">Ae. albopictus</span>, <span class="html-italic">Cx. pipiens</span> s.s./<span class="html-italic">Cx. torrentium</span>, <span class="html-italic">Ae. japonicus</span>, and <span class="html-italic">Cx. p. molestus</span>. The differences are indicated in red, with one of the species having a RCC below 1 indicating significant differences in behavior in a competitive environment.</p>
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<p>Mean viral RNA body titer per specimen and infection rate per species ratio. (<b>a</b>) Mean CHIKV RNA body titer per specimen of <span class="html-italic">Ae. albopictus</span>, (<b>b</b>) WNV infection rate of both <span class="html-italic">Cx. pipiens</span> strains, (<b>c</b>) mean WNV RNA body titer per specimen of <span class="html-italic">Cx. pipiens</span> s.s./<span class="html-italic">Cx. torrentium</span> (see Zenodo repository in data availability statement for separated results for bioforms) from the <span class="html-italic">Ae. albopictus</span> combination, (<b>d</b>) mean WNV RNA body titer per specimen of <span class="html-italic">Cx. p. molestus</span> from the <span class="html-italic">Ae. japonicus</span> combination. Infection rates were 100% and 0% for all <span class="html-italic">Ae. albopictus</span> and <span class="html-italic">Ae. japonicus</span> specimens, respectively; body titer was 0 genome copies per mosquito for <span class="html-italic">Ae. japonicus</span>. * = significant difference (<span class="html-italic">p</span> = 0.04).</p>
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<p>Infection Rates (IRs) of <span class="html-italic">Culex</span> species and <span class="html-italic">Cx. pipiens</span> biotypes used during the infection study.</p>
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<p>Principal Component Analysis (PCA) of total dataset with variables. Development time, larval ratio, mortality, cephalothorax length (CL), abdominal length (AL), abdominal width (AW), lipid (L), protein (P), glycogen (G), total distance moved, velocity, body contact, viral RNA body titer (BT), and infection rate (IR) for different species competition treatments in (<b>a</b>,<b>b</b>) <span class="html-italic">Ae. albopictus</span> and <span class="html-italic">Cx. pipiens</span> s.s./<span class="html-italic">Cx. torrentium</span> and (<b>c</b>,<b>d</b>) <span class="html-italic">Ae. japonicus</span> and <span class="html-italic">Cx. p. molestus</span> combinations at 26 °C. Infection rate is not included for <span class="html-italic">Ae. albopictus</span> and <span class="html-italic">Ae. japonicus</span>, viral RNA body titer is not included for <span class="html-italic">Ae. japonicus</span>.</p>
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10 pages, 1203 KiB  
Article
Formulation of a Commercial Quality Index for Avocado Produced in an Inter-Andean Valley
by John Peter Aguirre-Landa, Henrry Wilfredo Agreda-Cerna, David Quispe-Choque, Alfredo Prado-Canchari and Liliana Rodriguez Cardenas
Horticulturae 2024, 10(8), 783; https://doi.org/10.3390/horticulturae10080783 - 25 Jul 2024
Viewed by 497
Abstract
This study aimed to formulate a commercial quality index (CQI) for avocados (Persea americana Mill) produced in an inter-Andean valley in southern Peru. Thirty-eight commercial quality parameters of Hass and Fuerte avocados were evaluated under the marketing and export protocols approved in [...] Read more.
This study aimed to formulate a commercial quality index (CQI) for avocados (Persea americana Mill) produced in an inter-Andean valley in southern Peru. Thirty-eight commercial quality parameters of Hass and Fuerte avocados were evaluated under the marketing and export protocols approved in the Codex Alimentarius CXS 197-1995 issued by FAO and the Peruvian technical standard NTP 011.018. The index was formulated using information gathered from 44 experts in the Apurimac region. To weight the commercial quality parameters, the Delphi method was used, with the cooperation of expert producers and marketers, from which a weighted equation was formulated for the commercial quality index of Hass (CQIh) and Fuerte (CQIf) avocados. Fifteen parameters of interest were found for the formulation of the quality indexes for both varieties, which reported more than 50% coincidence among experts, based on physical and sensory evaluation. The CQI proposal would be a tool to help improve the quality attributes of avocado growers. Full article
(This article belongs to the Section Fruit Production Systems)
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<p>Fuerte and Hass Avocado.</p>
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<p>Avocado varieties’ producing areas.</p>
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20 pages, 1549 KiB  
Review
Fractalkine in Health and Disease
by Claudia Rodriguez, Luisa Chocarro, Miriam Echaide, Karina Ausin, David Escors and Grazyna Kochan
Int. J. Mol. Sci. 2024, 25(15), 8007; https://doi.org/10.3390/ijms25158007 - 23 Jul 2024
Viewed by 397
Abstract
CX3CL1 is one of the 50 up-to-date identified and characterized chemokines. While other chemokines are produced as small, secreted proteins, CX3CL1 (fractalkine) is synthetized as a transmembrane protein which also leads to a soluble form produced as a result of proteolytic cleavage. The [...] Read more.
CX3CL1 is one of the 50 up-to-date identified and characterized chemokines. While other chemokines are produced as small, secreted proteins, CX3CL1 (fractalkine) is synthetized as a transmembrane protein which also leads to a soluble form produced as a result of proteolytic cleavage. The membrane-bound protein and the soluble forms exhibit different biological functions. While the role of the fractalkine/CX3CR1 signaling axis was described in the nervous system and was also related to the migration of leukocytes to sites of inflammation, its actions are controversial in cancer progression and anti-tumor immunity. In the present review, we first describe the known biology of fractalkine concerning its action through its cognate receptor, but also its role in the activation of different integrins. The second part of this review is dedicated to its role in cancer where we discuss its role in anti-cancer or procarcinogenic activities. Full article
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<p>FKN polypeptides. Membrane-bound fractalkine can be shed by different proteases resulting in soluble forms. (<b>a</b>) Arrows show sites of proteolytic digestion and the proteases responsible for it. ADAM10, ADAM17, and CatS (blue); MMP2 (red); and secretases α, β, and γ (orange). Below the FKN full-length (FL) molecule, the collection of polypeptides generated via proteolytic cleavages are shown. The first and last amino acid residues are marked. (<b>b</b>) Function of FKN. The membrane-bound form (in blue) causes adhesion of leucocytes to the endothelial surface. It is also responsible for neuron–microglia crosstalk and survival (1). The soluble form (in blue) acts as chemoattractant for different cells which express its receptor CX3CR1 on their surface. Interaction with its receptor results in a signaling cascade (among others, integrin activation and inside-out activation mechanism, shown as colored serrated arrows). (2) Soluble fractalkine in the absence of its receptor CX3CR1 can bind to integrins in their inactive form (binding to the alternative site 2) and activate them (outside-in activation, shown as colored serrated arrows) that is followed by binding to their corresponding ligands (3).</p>
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<p>PDB structure of chemokine domain of FKN (PDB 1f2l). The chemokine domain of FKN in the crystal showed a quaternary arrangement [<a href="#B28-ijms-25-08007" class="html-bibr">28</a>]. In the figure, four FKN molecules are visible and are indicated in different colors. Nevertheless, FKN does not form oligomers in solution. However, it cannot be disregarded that, in the presence of other molecules, it may oligomerize.</p>
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<p>FKN in cancer immunotherapy. (<b>a</b>) <b>Top</b>: cancer cells (black) express PD-L1 on their surface that binds PD-L1 on the surface to T cells (blue cell on the left). This interaction inhibits T cell effector activities and creates a signaling barrier that protects cancer cells from interferon-induced apoptosis. <b>Bottom</b>: anti-PD-1 and anti-PD-L1 antibodies block PD-1/PD-L1 interactions, leading to enhanced T cell cytotoxicity towards cancer cells and increased sensitivity to interferon-induced apoptosis in cancer cells. (<b>b</b>) Plasma soluble FKN is a biomarker for immune cell diversity in peripheral blood from human NSCLC patients. <b>Left</b>: SPADE 3 hierarchical clustering of immune cell types in peripheral blood identified via high-dimensional flow cytometry in long-term responder patients to PD-1/PD-L1 blockade. <b>Right</b>: same as left but in non-responder patients [<a href="#B67-ijms-25-08007" class="html-bibr">67</a>]. The number of branches indicate the phenotypic diversity of immune cell types. Major lineages are grouped and indicated in the cluster trees. Mo, monocytes; NC-Mo, non-classical monocytes; G-MDSCs, granulocytic myeloid-derived suppressor cells; Neu, neutrophils. NK, natural killer cells. (<b>c</b>) FKN anti-tumor activities are dependent on NK and T cells. The graph shows a Kaplan–Meier survival plot in mice transplanted with lung cancer cells expressing soluble FKN, in which CD4, CD8, or NK cells have been depleted. FKN-dependent anti-tumor activities are eliminated following NK and T cell depletion [<a href="#B67-ijms-25-08007" class="html-bibr">67</a>]. **, indicates very significant differences by the Log-Rank test (<span class="html-italic">p</span> &lt; 0.01).</p>
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14 pages, 4223 KiB  
Article
The Central Conserved Peptides of Respiratory Syncytial Virus G Protein Enhance the Immune Response to the RSV F Protein in an Adenovirus Vector Vaccine Candidate
by Pengdi Chai, Yi Shi, Junjie Yu, Xiafei Liu, Dongwei Li, Jinsong Li, Lili Li, Dandi Li and Zhaojun Duan
Vaccines 2024, 12(7), 807; https://doi.org/10.3390/vaccines12070807 - 20 Jul 2024
Viewed by 772
Abstract
Respiratory syncytial virus (RSV) is a serious human respiratory pathogen that commonly affects children, older adults, and immunocompromised individuals. At present, the design of licensed vaccines focuses on the incorporation of the pre-fusion protein (PreF protein) of RSV, as this protein has the [...] Read more.
Respiratory syncytial virus (RSV) is a serious human respiratory pathogen that commonly affects children, older adults, and immunocompromised individuals. At present, the design of licensed vaccines focuses on the incorporation of the pre-fusion protein (PreF protein) of RSV, as this protein has the ability to induce antibodies that offer a high level of protection. Moreover, the G protein contains the CX3C motif that binds the chemokine receptor CX3CR1 in respiratory epithelial cells, which plays an essential role in viral infection. Therefore, incorporating the G antigen into vaccine design may prove more advantageous for RSV prevention. In this study, we developed a human adenoviral vector-based RSV vaccine containing highly neutralizing immunogens, a modified full-length PreF protein fused with the central conserved peptides of the G protein (Gcc) from both RSV subgroups trimerized via a C-terminal foldon, and evaluated its immune response in mice through intranasal (i.n.) immunization. Our results showed that immunization with Ad5-PreF-Qa-Gcc elicited a balanced Th1/Th2 immune response and robust mucosal immunity with higher neutralizing antibody titers against RSV Long and RSV B1. Importantly, immunization with Ad5-PreF-Qa-Gcc enhanced CD4+ CD25+ FoxP3+ Treg cell response and protected the mice against RSV infection. Our data demonstrate that the combination of Gcc and the PreF antigen is a viable strategy for developing effective RSV vaccines. Full article
(This article belongs to the Special Issue Recent Developments in Vaccines against Respiratory Pathogens)
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<p>Designing and characterizing the Ad-based vaccines. (<b>a</b>) Schematic of Ad5-PreF, Ad5-Gcc, Ad5-PreF-P2A-Gcc and Ad5-PreF-Qa-P2A-Gcc. (<b>b</b>,<b>c</b>) Western blot analysis of F expression in cell lysis (<b>b</b>) and supernatant (<b>c</b>). (<b>d</b>,<b>e</b>) Western blot analysis of Gcc expression in cell lysis (<b>d</b>) and supernatant (<b>e</b>). (<b>f</b>) Identification of the conformation of F protein. Ad5-empty was used as control. Site Ø represents the unique antigenic sites of PreF.</p>
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<p>Immune responses in immunized Balc/c mice. (<b>a</b>) Schedule of Ad5 vaccines immunization and bleeding strategies in female Balb/C mice. (<b>b</b>) Kinetics of PreF-specific total IgG reciprocal endpoint titers (log10) were measured within 8 weeks of initial vaccination. (<b>c</b>) Kinetics of G-specific total IgG reciprocal endpoint titers (log10) were measured within 8 weeks after vaccination. (<b>d</b>) Endpoint titer of PreF-specific total IgG was measured in serum at week 6. (<b>e</b>) Endpoint titer of PreF-specific total IgA was measured in serum at week 6. (<b>f</b>) Endpoint titer of G-specific total IgG was measured in serum at week 6. (<b>g</b>) Endpoint titer of G-specific total IgA was measured in serum at week 6. (<b>h</b>) Endpoint titer of PreF-specific total IgG was measured in BALF at week 8. (<b>i</b>) Endpoint titer of PreF-specific total IgA was measured in BALF at week 8. (<b>j</b>) Endpoint titer of G-specific total IgG was measured in BALF at week 8. (<b>k</b>) Endpoint titer of G-specific total IgA was measured in BALF at week 8. ns = not significant; * <span class="html-italic">p</span> &lt; 0.05, **** <span class="html-italic">p</span> &lt; 0.0001.</p>
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<p>Intranasal immunization induced a balanced Th1/Th2 immune response in mice vaccinated with Ad-PreF-Qa-P2A-Gcc. (<b>a</b>–<b>c</b>) Anti-PreF antibody subtype IgG1 (<b>a</b>) and IgG2a (<b>b</b>) were detected in serum at week 6, and the ratio of IgG2a/IgG1 (<b>c</b>) was calculated. (<b>d</b>–<b>f</b>) Anti-G antibody subtypes IgG1 (<b>d</b>) and IgG2a (<b>e</b>) were detected in serum at week 6, and the ratio of IgG2a/IgG1 (<b>f</b>) was calculated. ns = not significant; *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Mice vaccinated with Ad5-PreF-Qa-Gcc induced superior neutralizing antibody. (<b>a</b>,<b>b</b>) Serum-neutralizing antibody response against RSV Long (<b>a</b>) and RSV B1 (<b>b</b>) at week 6. All data are shown as means ± SEM. <span class="html-italic">p</span>-values were analyzed with one-way ANOVA (* <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).</p>
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<p>Immunogenicity and protective efficacy in BALB/c mice. (<b>a</b>) Schematics of vaccine immunization and challenge in BALB/c mice (6–8 weeks old). Mice were challenged with 2 × 10<sup>6</sup> pfu RSV Long via the i.n. route at 4 weeks after boost vaccination. (<b>b</b>) Body weight change over 5 days post-infection. (<b>c</b>,<b>d</b>) Copies of RSV viral L gene in lung tissue (<b>c</b>) and nasal tissue (<b>d</b>). The limit of detection for the RT-qPCRs is 10 copies/mL. (<b>e</b>–<b>j</b>) Tissue sections of the lung tissues were stained with H&amp;E and PAS for pathological examination. The magnification of all images is 20x. All data are shown as means ± SEM. <span class="html-italic">p</span>-values were analyzed with one-way ANOVA (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001).</p>
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<p>Treg cells and Th17 cells in the lung of vaccinated mice induced by RSV challenge infection. Vaccinated mice were challenged i.n. with RSV Long at 6 weeks after the immunization. (<b>a</b>) The percentage of CD4<sup>+</sup>CD25<sup>+</sup>Foxp3<sup>+</sup> Treg cells. (<b>b</b>) The percentage of IL-17A<sup>+</sup> CD4<sup>+</sup> T cells. All data are shown as means ± SEM. <span class="html-italic">p</span>-values were analyzed with one-way ANOVA (** <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).</p>
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25 pages, 8864 KiB  
Article
A Real-Time and Privacy-Preserving Facial Expression Recognition System Using an AI-Powered Microcontroller
by Jiajin Zhang, Xiaolong Xie, Guoying Peng, Li Liu, Hongyu Yang, Rong Guo, Juntao Cao and Jianke Yang
Electronics 2024, 13(14), 2791; https://doi.org/10.3390/electronics13142791 - 16 Jul 2024
Viewed by 652
Abstract
This study proposes an edge computing-based facial expression recognition system that is low cost, low power, and privacy preserving. It utilizes a minimally obtrusive cap-based system designed for the continuous and real-time monitoring of a user’s facial expressions. The proposed method focuses on [...] Read more.
This study proposes an edge computing-based facial expression recognition system that is low cost, low power, and privacy preserving. It utilizes a minimally obtrusive cap-based system designed for the continuous and real-time monitoring of a user’s facial expressions. The proposed method focuses on detecting facial skin deformations accompanying changes in facial expressions. A multi-zone time-of-flight (ToF) depth sensor VL53L5CX, featuring an 8 × 8 depth image, is integrated into the front brim of the cap to measure the distance between the sensor and the user’s facial skin surface. The distance values corresponding to seven universal facial expressions (neutral, happy, disgust, anger, surprise, fear, and sad) are transmitted to a low-power STM32F476 microcontroller (MCU) as an edge device for data preprocessing and facial expression classification tasks utilizing an on-device pre-trained deep learning model. Performance evaluation of the system is conducted through experiments utilizing data collected from 20 subjects. Four deep learning algorithms, including Convolutional Neural Networks (CNN), Recurrent Neural Networks (RNN), Long Short-Term Memory (LSTM) networks, and Deep Neural Networks (DNN), are assessed. These algorithms demonstrate high accuracy, with CNN yielding the best result, achieving an accuracy of 89.20% at a frame rate of 15 frames per second (fps) and a maximum latency of 2 ms. Full article
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<p>System overview, showing the hardware connections and data processing pipeline.</p>
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<p>VL53L5CX sensor.</p>
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<p>Facial muscle distances matrix captured by the sensor (8 × 8 Configuration).</p>
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<p>The facial skin surface detection area of the VL53L5CX.</p>
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<p>SensorTile kit.</p>
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<p>Cap with hardware distribution.</p>
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<p>Depth images for facial expressions. A camera image (<b>left</b>) and a corresponding ToF depth image (<b>right</b>).</p>
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<p>Depth images for facial expressions. A camera image (<b>left</b>) and a corresponding ToF depth image (<b>right</b>).</p>
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<p>Experimental setting.</p>
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<p>Structure of the CNN model used for classification.</p>
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<p>Structure of the Simple RNN model used for classification.</p>
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<p>Structure of the LSTM model used for classification.</p>
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<p>Structure of the DNN model used for classification.</p>
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<p>Deep learning model quantization and porting.</p>
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<p>Accuracy graphs of the validation datasets.</p>
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<p>Loss of the validation datasets.</p>
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<p>CNN model AUC-ROC curve.</p>
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<p>RNN model AUC-ROC curve.</p>
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<p>LSTM model AUC-ROC curve.</p>
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<p>DNN model AUC-ROC curve.</p>
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<p>Confusion matrix generated using the CNN algorithm.</p>
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<p>Confusion matrix generated using the RNN algorithm.</p>
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<p>Confusion matrix generated using the LSTM algorithm.</p>
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<p>Confusion matrix generated using the DNN algorithm.</p>
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<p>Screenshots of smartphone application. (<b>a</b>) Recognition page; (<b>b</b>) Recording page.</p>
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<p>Watching films.</p>
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<p>Riding a bus.</p>
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<p>Walking.</p>
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12 pages, 6521 KiB  
Article
Novel Superhard Tetragonal Hybrid sp3/sp2 Carbon Allotropes Cx (x = 5, 6, 7): Crystal Chemistry and Ab Initio Studies
by Samir F. Matar and Vladimir L. Solozhenko
C 2024, 10(3), 64; https://doi.org/10.3390/c10030064 - 16 Jul 2024
Viewed by 524
Abstract
Novel superhard tetragonal carbon allotropes C5, C6, and C7, characterized by the presence of sp3- and sp2-like carbon sites, have been predicted from crystal chemistry and extensively studied by quantum density functional theory [...] Read more.
Novel superhard tetragonal carbon allotropes C5, C6, and C7, characterized by the presence of sp3- and sp2-like carbon sites, have been predicted from crystal chemistry and extensively studied by quantum density functional theory (DFT) calculations. All new allotropes were found to be cohesive, with crystal densities and cohesive energies decreasing along the C5-C6-C7 series due to the greater openness of the structures resulting from the presence of C=C ethene and C=C=C propadiene subunits, and they were mechanically stable, with positive sets of elastic constants. The Vickers hardness evaluated by different models qualifies all allotropes as superhard, with Hv values ranging from 90 GPa for C5 to 79 GPa for C7. Phonon band structures confirm that the new allotropes are also dynamically stable. The electronic band structures reveal their metallic-like behavior due to the presence of sp2-hybridized carbon. Full article
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<p>From <span class="html-italic">tet</span>-C<sub>4</sub> (<b>a</b>) to <span class="html-italic">tet</span>-C<sub>5</sub> (<b>b</b>) by changing the atomic positions. The carbon atoms numbers correspond to the numbering in <a href="#carbon-10-00064-t001" class="html-table">Table 1</a>.</p>
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<p>Crystal structures of new tetragonal carbon allotropes in three representations: ball-and-stick (<b>left</b>), polyhedral (<b>middle</b>), and double cells with numbering of atoms as in <a href="#carbon-10-00064-t002" class="html-table">Table 2</a>: C<sub>5</sub> (<b>a</b>); C<sub>6</sub> (<b>b</b>); C<sub>7</sub> (<b>c</b>).</p>
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<p>Simulated X-ray diffraction patterns of new tetragonal carbon allotropes.</p>
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<p>Charge density projections of tetragonal C<sub>5</sub> (<b>a</b>); C<sub>6</sub> (<b>b</b>); and C<sub>7</sub> (<b>c</b>).</p>
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<p>Calculated total energy per atom as a function of volume for new tetragonal carbon allotropes and diamond.</p>
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<p>Phonon band structures of new carbon allotropes along the major directions of the simple tetragonal Brillouin zone: C<sub>5</sub> (<b>a</b>); C<sub>6</sub> (<b>b</b>); C<sub>7</sub> (<b>c</b>).</p>
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<p>Heat capacities at constant volume (C<sub>V</sub>) (<b>a</b>) and entropies (S) (<b>b</b>) of new tetragonal carbon allotropes compared to diamond. Experimental C<sub>V</sub> data of diamond [<a href="#B38-carbon-10-00064" class="html-bibr">38</a>,<a href="#B39-carbon-10-00064" class="html-bibr">39</a>] are shown as gray symbols.</p>
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<p>Electronic band structures of new tetragonal carbon allotropes: C<sub>5</sub> (<b>a</b>); C<sub>6</sub> (<b>b</b>); C<sub>7</sub> (<b>c</b>); the tetragonal Brillouin zone (<b>d</b>).</p>
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8 pages, 1416 KiB  
Proceeding Paper
A Systematic Study on the Effect of the Xanthation Temperature on Viscose Quality
by Novita Novita, Budhijanto Budhijanto and Muhammad Mufti Azis
Eng. Proc. 2024, 67(1), 6; https://doi.org/10.3390/engproc2024067006 - 16 Jul 2024
Viewed by 182
Abstract
The xanthation reaction is an exothermic reaction between alkali cellulose (AC) and carbon disulfide (CS2) used to regenerate a viscose solution. The cooling system plays an important role during the reaction to yield more of the main product, cellulose xanthate (CX), [...] Read more.
The xanthation reaction is an exothermic reaction between alkali cellulose (AC) and carbon disulfide (CS2) used to regenerate a viscose solution. The cooling system plays an important role during the reaction to yield more of the main product, cellulose xanthate (CX), instead of the by-product, sodium trithiocarbonate (TTC). Minimizing the yield of by-products during the reaction phase may lead to less by-product generation during the ripening process due to free caustic and excess CS2 in the system. The reaction was performed in a batch reactor with an agitator (9.7 rpm) under vacuum conditions (350 mbar), and the temperature varied from 20 °C to 35 °C, as is applicable in industrial plants. Meanwhile, the CX and TTC were determined via UV spectroscopy. Since the temperature reaction will affect the period of the reaction, which impacts the productivity of industrial applications, the experiment was conducted with a temperature change during the reaction to obtain a good-quality product without impacting productivity. This work aimed to reach an optimum xanthation temperature under the same combination of hardwood and softwood dissolving pulp. The results indicated that the xanthation reaction has an advantage at lower temperatures compared to higher ones; however, having a lower temperature led to a longer reaction period. The TTC was shown to be 17.7% lower at lower temperatures than at higher temperatures, which means that the CX was at a higher percentage at lower temperatures. Interestingly, the combination of higher and lower temperatures gave good viscose quality, which may lead to less consumption of CS2 and improve the environment due to less sulfur production during spinning. Full article
(This article belongs to the Proceedings of The 3rd International Electronic Conference on Processes)
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<p>Product (CX) content at different xanthation temperatures and process stages based on laboratory results.</p>
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<p>By-product (TTC) content at different xanthation temperatures and process stages based on laboratory results.</p>
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<p>Total pressure as a function of the xanthation period at different xanthation temperatures based on laboratory results.</p>
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13 pages, 2367 KiB  
Article
Silmitasertib (CX-4945) Disrupts ERα/HSP90 Interaction and Drives Proteolysis through the Disruption of CK2β Function in Breast Cancer Cells
by Hogyoung Kim, Emma Elkins, Rahib Islam, Bo Cao, Nour Abbes, Kaela Battles, Sihyoung Kim, Sichan Kim and Christopher Williams
Cancers 2024, 16(14), 2501; https://doi.org/10.3390/cancers16142501 - 10 Jul 2024
Viewed by 614
Abstract
Aberrant estrogen receptor (ERα) signaling mediates detrimental effects of tamoxifen including drug resistance and endometrial hyperplasia. ERα36, an alternative isoform of ERα, contributes to these effects. We have demonstrated that CK2 modulates ERα expression and function in breast cancer (BCa). Here, we assess [...] Read more.
Aberrant estrogen receptor (ERα) signaling mediates detrimental effects of tamoxifen including drug resistance and endometrial hyperplasia. ERα36, an alternative isoform of ERα, contributes to these effects. We have demonstrated that CK2 modulates ERα expression and function in breast cancer (BCa). Here, we assess if CX-4945 (CX), a clinical stage CK2 inhibitor, can disrupt ERα66 and ERα36 signaling in BCa. Using live cell imaging, we assessed the antiproliferative effects of CX in tamoxifen-sensitive and tamoxifen-resistant BCa cells in monolayer and/or spheroid cultures. CX-induced alterations in ERα66 and ERα36 mRNA and protein expression were assessed by RT-PCR and immunoblot. Co-immunoprecipitation was performed to determine the differential interaction of ERα isoforms with HSP90 and CK2 upon CX exposure. CX caused concentration-dependent decreases in proliferation in tamoxifen-sensitive MCF-7 and tamoxifen-resistant MCF-7 Tam1 cells and significantly repressed spheroid growth in 3D models. Additionally, CX caused dramatic decreases in endogenous or exogenously expressed ERα66 and ERα36 protein. Silencing of CK2β, the regulatory subunit of CK2, resulted in destabilization and decreased proliferation, similar to CX. Co-immunoprecipitation demonstrated that ERα66/36 show CK2 dependance for interaction with molecular chaperone HSP90. Our findings show that CK2 functions regulate the protein stability of ERα66 and ERα36 through a mechanism that is dependent on CK2β subunit and HSP90 chaperone function. CX may be a component of a novel therapeutic strategy that targets both tamoxifen-sensitive and tamoxifen-resistant BCa, providing an additional tool to treat ERα-positive BCa. Full article
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<p>Temporal and concentration-dependent effects of CX on BCa cells. Live cell imaging of cell confluence with exposure of MCF-7 (<b>A</b>,<b>B</b>) to CX or 4-OHT, respectively. MCF-7 Tam1 cells (<b>C</b>,<b>D</b>) were treated as described in (<b>A</b>,<b>B</b>). (<b>E</b>) MCF-7 tumor spheroids were exposed to CX for up to 15 days and spheroid diameter monitored (scale bar: 300 μm). Statistical analysis was performed by 2-way ANOVA or mixed effects, using Dunnett’s multiple comparison’s post hoc test, where * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01, *** <span class="html-italic">p</span> ≤ 0.005, **** <span class="html-italic">p</span> ≤ 0.001 and compared with the control group.</p>
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<p>Effects of CX on ERα66 and ERα36 expression in tamoxifen-sensitive and -resistant MCF-7 cells. (<b>A</b>) ERα66 and ERα36 RNA transcript levels in MCF-7 and MCF-7 Tam1 cells were measured by qRT-PCR. (<b>B</b>) ERα66 and ERα36 protein levels in MCF-7 and MCF-7 Tam1 cells were determined by western immunoblot. (<b>C</b>) MCF-7 Tam1 cells were transfected with a scrambled or ERα36-targeting siRNA, and subsequently assessed ERα66/36 protein levels by immunoblot. (<b>D</b>) ERα66 and ERα36 RNA transcript levels in MCF-7 and MCF-7 Tam1 cells were measured by qRT-PCR, following 24 h CX exposure at the indicated doses. ERα66 and/or ERα36 protein levels in MCF-7 (<b>E</b>), MCF-7 Tam1 (<b>F</b>), MDA-MB-231 (ERα36 only, (<b>G</b>)) and T47-D (<b>H</b>) cells by Western immunoblot following 24 h CX exposure at the indicated concentrations. (<b>I</b>) The impact of CX on the expression of ER66/36 levels in the presence and absence of 4-OHT in MCF-7 cells by immunoblot. Ratiometric analysis was performed to assess ERα66/36 changes relative to control and normalized to β-actin expression. For qRT-PCR, statistical analysis was performed by 2-way ANOVA using Dunnett’s multiple comparison’s post hoc test, where * <span class="html-italic">p</span> ≤ 0.05 and ** <span class="html-italic">p</span> ≤ 0.01 compared with the control group. Full blots are presented in <a href="#app1-cancers-16-02501" class="html-app">Supplementary Figure S4</a>.</p>
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<p>Role of proteolysis in CX-mediated downregulation of ERα66/36. (<b>A</b>) HEK293T cells stably transfected with mGFP, ERα66-mGFP, or ERα36-mGFP. Immunoblot using mGFP antibodies were performed. The panel shows mGFP immunoreactivity of each cell line. (<b>B</b>) 293T-mGFP, 293T-ERα66-mGFP, and 293T-ERα36-mGFP cells were exposed to CX for 24hr. mGFP and β-actin expression were ascertained by immunoblot. (<b>C</b>) MCF-7 and MCF-7 Tam1 cells were exposed to CX in the presence or absence of protein synthesis inhibitor cycloheximide (CHX) and proteosome inhibitor MG132. ERα66/36, HSP90, GAPDH, and β-actin expression were ascertained by immunoblot. For WB, statistical analysis was performed by 2-way ANOVA using Dunnett’s multiple comparison’s post hoc test, where * <span class="html-italic">p</span> ≤ 0.05 and compared with the control group. Full blots are presented in <a href="#app1-cancers-16-02501" class="html-app">Supplementary Figure S4</a>.</p>
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<p>CX exposure impacts CK2β subunit phosphorylation and expression. (<b>A</b>) Expression of CK2 subunits (α, α’, β) in MCF-7 and MCF-7 Tam1 by qRT-PCR. (<b>B</b>) α, α’, and β subunit protein levels in MCF-7 and MCF-7 Tam1 cells were determined by immunoblot, and β-actin. (<b>C</b>) α, α’, β subunit, and S209 phosphorylation of CK2β protein level in MCF-7 and MCF-7 Tam1 cells with 5 µM CX by western immunoblot following 24 h. (<b>D</b>) of β subunit expression in MCF-7 and MCF-7 Tam1 with CX by qRT-PCR following 10 or 24 h. (<b>E</b>) Co-immunoprecipitation of HSP90, ERα, and CK2β in MCF-7 Tam1 cells after 4 h exposure to 5 µM CX. (<b>F</b>) ERα/HSP90 signal measured by flow-PLA for MCF-7 cells treated with CX. The stimulated cells without the corresponding target binding probes served as the control. Data represent of 3 independent experiments. (<b>G</b>) MCF-7 and MCF-7 Tam1 cells were treated with control or CK2β shRNA. The transduced cells were selected with puromycin dihydrochloride. Protein lysates were analyzed for ERα66, ERα36, and CK2β, expression by Western blotting. (<b>H</b>) Incucyte<sup>®</sup> proliferation assay analysis of MCF-7 cells seeded at 5 × 10<sup>3</sup> cells/well in a 96-well plate. The cells were transfected with a shRNA control or targeting CK2β. For qRT-PCR and WB, statistical analysis was performed by 2-way ANOVA using Dunnett’s multiple comparison’s post hoc test, where * <span class="html-italic">p</span> ≤ 0.05 and ** <span class="html-italic">p</span> ≤ 0.01 compared with the control group. Full blots are presented in <a href="#app1-cancers-16-02501" class="html-app">Supplementary Figure S4</a>.</p>
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<p>Model of CK2 regulated ERα stability in BCa. Auto-phosphorylated CK2 phosphorylates ERα66, ERα36, and HSP90, resulting in increased association and stability of the estrogen receptors. Inhibition by CX-4945 results in reduced phosphorylation of the substrates, resulting in enhanced degradation of both ERα isoforms.</p>
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22 pages, 3132 KiB  
Article
Neurosteroid [3α,5α]-3-Hydroxy-pregnan-20-one Enhances the CX3CL1-CX3CR1 Pathway in the Brain of Alcohol-Preferring Rats with Sex-Specificity
by Irina Balan, Adelina Grusca, Samantha Lucenell Chéry, Baylee R. Materia, Todd K. O’Buckley and A. Leslie Morrow
Life 2024, 14(7), 860; https://doi.org/10.3390/life14070860 - 9 Jul 2024
Cited by 1 | Viewed by 550
Abstract
This study investigates the impact of allopregnanolone ([3α,5α]3-hydroxypregnan-20-one or 3α,5α-tetrahydroprogesterone (3α,5α-THP); 10 mg/kg, IP) on fractalkine/CX3-C motif chemokine ligand 1 (CX3CL1) levels, associated signaling components, and markers for microglial and astrocytic cells in the nucleus accumbens (NAc) of male and female alcohol-preferring (P) [...] Read more.
This study investigates the impact of allopregnanolone ([3α,5α]3-hydroxypregnan-20-one or 3α,5α-tetrahydroprogesterone (3α,5α-THP); 10 mg/kg, IP) on fractalkine/CX3-C motif chemokine ligand 1 (CX3CL1) levels, associated signaling components, and markers for microglial and astrocytic cells in the nucleus accumbens (NAc) of male and female alcohol-preferring (P) rats. Previous research suggested that 3α,5α-THP enhances anti-inflammatory interleukin-10 (IL-10) cytokine production in the brains of male P rats, with no similar effect observed in females. This study reveals that 3α,5α-THP elevates CX3CL1 levels by 16% in the NAc of female P rats, with no significant changes observed in males. The increase in CX3CL1 levels induced by 3α,5α-THP was observed in females across multiple brain regions, including the NAc, amygdala, hypothalamus, and midbrain, while no significant effect was noted in males. Additionally, female P rats treated with 3α,5α-THP exhibited notable increases in CX3CL1 receptor (CX3CR1; 48%) and transforming growth factor-beta 1 (TGF-β1; 24%) levels, along with heightened activation (phosphorylation) of signal transducer and activator of transcription 1 (STAT1; 85%) in the NAc. Conversely, no similar alterations were observed in male P rats. Furthermore, 3α,5α-THP decreased glial fibrillary acidic protein (GFAP) levels by 19% in both female and male P rat NAc, without affecting microglial markers ionized calcium-binding adaptor molecule 1 (IBA1) and transmembrane protein 119 (TMEM119). These findings indicate that 3α,5α-THP enhances the CX3CL1/CX3CR1 pathway in the female P rat brain but not in males, primarily influencing astrocyte reactivity, with no observed effect on microglial activation. Full article
(This article belongs to the Section Physiology and Pathology)
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<p>(<b>a</b>) 3α,5α-THP upregulates the levels of CX3-C motif chemokine ligand 1/fractalkine (CX3CL1) in the nucleus accumbens (NAc) of female, but not male alcohol-preferring (P) rats. Male and female P rats (n = 10/group) were treated intraperitoneally with 3α,5α-THP (10 mg/kg) or vehicle (45% <span class="html-italic">w</span>/<span class="html-italic">v</span> 2-hydroxypropyl-β-cyclodextrin) control. After 60 min, the nucleus accumbens (NAc) was examined using ELISA to determine CX3CL1 expression. In females, the administration of 3α,5α-THP resulted in a significant increase in CX3CL1 levels within the NAc (<span class="html-italic">p</span> = 0.02). However, in male rats, 3α,5α-THP treatment did not lead to a notable change in CX3CL1 expression within the NAc (<span class="html-italic">p</span> = 0.91). In the graphs, every column, along with its error bar, represents the mean ± SEM, expressed in pg/mg of total protein level. Each circle represents an individual CX3CL1 value for vehicle-treated rats, while the black squares indicate the CX3CL1 values for the 3α,5α-THP-treated rats. * <span class="html-italic">p</span> &lt; 0.05. (<b>b</b>) Qualitative evaluation of 3α,5α-THP’s impact on the intracellular distribution of CX3CL1 in the NAc of female P rats. Double-immunofluorescent staining was conducted using antibodies targeting CX3CL1 alongside NeuN (a neuronal marker), TMEM119 (a microglial marker) or GFAP (an astrocyte marker). In vehicle control, CX3CL1 was observed to localize within NeuN-positive neuronal cells while not co-localizing with TMEM119-positive microglial cells or GFAP-positive astrocytic cells. Treatment with 3α,5α-THP did not induce any evident alterations in CX3CL1’s intracellular localization. Scale bar is 50 µm.</p>
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<p>3α,5α-THP enhances CX3CR1 levels in the nucleus accumbens (NAc) of female P rats, but not in males. Male and female alcohol-preferring (P) rats (n = 10/group) were treated intraperitoneally with 3α,5α-THP (10 mg/kg) or vehicle (45% <span class="html-italic">w</span>/<span class="html-italic">v</span> 2-hydroxypropyl-β-cyclodextrin) control. After 60 min, the NAc was examined using immunoblotting assays to determine CX3CR1 expression. Specifically, in females, administering 3α,5α-THP resulted in a significant elevation of CX3CR1 levels within the NAc (<span class="html-italic">p</span> = 0.0006). Conversely, in male rats, treatment with 3α,5α-THP did not induce a noteworthy alteration in CX3CR1 expression within the NAc (<span class="html-italic">p</span> = 0.98). In the graphs, every column, along with its error bar, represents the mean ± SEM, expressed as a percentage relative to the average value of the vehicle control. Each circle represents an individual CX3CR1 value, normalized to β-Actin for vehicle-treated rats, while the black squares indicate the corresponding values for the 3α,5α-THP-treated rats. *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>3α,5α-THP upregulates the levels of pSTAT1 in the nucleus accumbens (NAc) of female, but not male P rats. Male and female alcohol-preferring (P) rats (n = 10/group) were treated intraperitoneally with 3α,5α-THP (10 mg/kg) or vehicle (45% <span class="html-italic">w</span>/<span class="html-italic">v</span> 2-hydroxypropyl-β-cyclodextrin) control. After 60 min, the NAc was examined using immunoblotting assays to determine pSTAT1 expression. In females, the administration of 3α,5α-THP resulted in a significant increase in pSTAT1 levels within the NAc (<span class="html-italic">p</span> = 0.0002). However, in male rats, 3α,5α-THP treatment did not lead to a notable change in pSTAT1 expression within the NAc (<span class="html-italic">p</span> = 0.08). In the graphs, every column, along with its error bar, represents the mean ± SEM, expressed as a percentage relative to the average value of the vehicle control. Each circle represents an individual pSTAT1 value, normalized to β-Actin for vehicle-treated rats, while the black squares indicate the corresponding values for the 3α,5α-THP-treated rats. *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>3α,5α-THP upregulates the levels of TGF-β1 in the nucleus accumbens (NAc) of female, but not male P rats. Male and female alcohol-preferring (P) rats (n = 10/group) were treated intraperitoneally with 3α,5α-THP (10 mg/kg) or vehicle (45% <span class="html-italic">w</span>/<span class="html-italic">v</span> 2-hydroxypropyl-β-cyclodextrin) control. After 60 min, the NAc was examined using immunoblotting assays to determine TGF-β1 expression. In females, the administration of 3α,5α-THP (10 mg/kg, IP) resulted in a significant increase in TGF-β1 levels within the NAc (<span class="html-italic">p</span> = 0.04). However, in male rats, 3α,5α-THP treatment did not lead to a notable change in TGF-β1 expression within the NAc (<span class="html-italic">p</span> = 0.21). In the graphs, every column, along with its error bar, represents the mean ± SEM, expressed as a percentage relative to the average value of the vehicle control. Each circle represents an individual TGF-β1 value, normalized to β-Actin for vehicle-treated rats, while the black squares indicate the corresponding values for the 3α,5α-THP-treated rats. * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>3α,5α-THP downregulates the levels of the astrocytic marker GFAP in the nucleus accumbens (NAc) of female and male P rats. Male and female alcohol-preferring (P) rats (n = 10/group) were treated intraperitoneally with 3α,5α-THP (10 mg/kg) or vehicle (45% <span class="html-italic">w</span>/<span class="html-italic">v</span> 2-hydroxypropyl-β-cyclodextrin) control. After 60 min, the NAc was examined using immunoblotting assays to determine GFAP expression. In both females (<span class="html-italic">p</span> = 0.02) and males (<span class="html-italic">p</span> = 0.03), administering 3α,5α-THP led to a significant decrease in GFAP levels within the NAc. In the graphs, every column, along with its error bar, represents the mean ± SEM, expressed as a percentage relative to the average value of the vehicle control. Each circle represents an individual GFAP value, normalized to β-Actin for vehicle-treated rats, while the black squares indicate the corresponding values for the 3α,5α-THP-treated rats. * <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Schematic of 3α,5α-THP (allopregnanolone) actions on CX3CL1/CX3CR1 signaling in the brain of female ethanol-naïve alcohol-preferring (P) rats. Initially, activation of inflammatory TLR pathways leads to the upregulation of inflammatory factors, resulting in decreased levels (red arrows down) of CX3CL1 in neurons and CX3CR1 in glia, thereby reducing (black double-ended arrows crossed by a red zigzag) CX3CL1/CX3CR1 signaling between neurons and glia. Concurrently, GFAP upregulation (red arrow up) in astrocytes also contributes to disrupted neuron–glial communication. Upon administration of 3α,5α-THP, inflammatory TLR pathways are inhibited, resulting in the downregulation of inflammatory factors. Subsequently, CX3CL1 levels in neurons increase (green arrow up) through the activation (phosphorylation) of STAT1 (pSTAT1 upregulation is represented by a green arrow up), while CX3CR1 levels in glia are upregulated (green arrow up) via TGF-β1 (TGF-β1 upregulation is represented by a green arrow up) involvement. Additionally, GFAP levels in astrocytes decrease (green arrow down). These changes ultimately lead to enhanced neuron–glial communication (black double-ended arrows). Created with BioRender.com.</p>
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21 pages, 1410 KiB  
Review
Chemokine CX3CL1 (Fractalkine) Signaling and Diabetic Encephalopathy
by Mateusz Wątroba, Anna D. Grabowska and Dariusz Szukiewicz
Int. J. Mol. Sci. 2024, 25(14), 7527; https://doi.org/10.3390/ijms25147527 (registering DOI) - 9 Jul 2024
Viewed by 518
Abstract
Diabetes mellitus (DM) is the most common metabolic disease in humans, and its prevalence is increasing worldwide in parallel with the obesity pandemic. A lack of insulin or insulin resistance, and consequently hyperglycemia, leads to many systemic disorders, among which diabetic encephalopathy (DE) [...] Read more.
Diabetes mellitus (DM) is the most common metabolic disease in humans, and its prevalence is increasing worldwide in parallel with the obesity pandemic. A lack of insulin or insulin resistance, and consequently hyperglycemia, leads to many systemic disorders, among which diabetic encephalopathy (DE) is a long-term complication of the central nervous system (CNS), characterized by cognitive impairment and motor dysfunctions. The role of oxidative stress and neuroinflammation in the pathomechanism of DE has been proven. Fractalkine (CX3CL1) has unique properties as an adhesion molecule and chemoattractant, and by acting on its only receptor, CX3CR1, it regulates the activity of microglia in physiological states and neuroinflammation. Depending on the clinical context, CX3CL1-CX3CR1 signaling may have neuroprotective effects by inhibiting the inflammatory process in microglia or, conversely, maintaining/intensifying inflammation and neurotoxicity. This review discusses the evidence supporting that the CX3CL1-CX3CR1 pair is neuroprotective and other evidence that it is neurotoxic. Therefore, interrupting the vicious cycle within neuron–microglia interactions by promoting neuroprotective effects or inhibiting the neurotoxic effects of the CX3CL1-CX3CR1 signaling axis may be a therapeutic goal in DE by limiting the inflammatory response. However, the optimal approach to prevent DE is simply tight glycemic control, because the elimination of dysglycemic states in the CNS abolishes the fundamental mechanisms that induce this vicious cycle. Full article
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<p>Schematic structure of the C-X3-C motif chemokine ligand 1 (CX3CL1), also known as fractalkine or neurotactin. Both forms of the chemokine are shown: membrane-bound (transmembrane) CX3CL1 and soluble CX3CL1. The soluble CX3CL1, containing an N-terminal chemokine domain and an extracellular mucin-like stalk, is generated through the cleavage of the membrane-bound molecule near the outer surface of the membrane (marked symbolically with scissors and crossed red lines). Release of soluble CX3CL1 may occur upon exposure to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), tumor necrosis factor alpha (TNF-α) converting enzyme (TACE or ADAM17), matrix metalloproteinase-2 (MMP-2), or cathepsins (CTS). Adapted from [<a href="#B63-ijms-25-07527" class="html-bibr">63</a>].</p>
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<p>Downstream signaling pathways (A–D) after the activation of the CX3C motif chemokine receptor 1 (CX3CR1) caused by the attachment of the only endogenous ligand, chemokine CX3CL1 (fractalkine), released in a soluble form (sCX3CL1) from neurons. Gene transcription changes are a consequence of the signal transducer and activator of the transcription protein (STAT), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and cAMP/Ca<sup>2+</sup> response element binding protein (CREB) activation, while inhibiting the members of the class O of forkhead box transcription factors (FOXO). To keep the figure clear, the interaction of CX3CR1 with other receptors has been omitted.</p>
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<p>The importance of microglia and CX3CL1/CX3CR1 signaling in the vicious cycle of microglia–neuron interaction during neuroinflammation in diabetic encephalopathy. Microglia activation in diabetic encephalopathy occurs as a result of the action of signaling pathways related to the following: ❶ pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), triggering receptor expressed on myeloid cells 2/DNAX-activating protein of 12 kDa (TRM2/DAP12), and advanced glycation end product/receptor for advanced glycation end product (AGE-RAGE), ❷ the activation of p38 mitogen-activated protein kinases, a class of mitogen-activated protein kinases (MAPKs) p38 MAPK, and ❸ purinergic signaling. Microglial inflammation may also develop as a consequence of blocking the interaction of the immunomodulatory protein CD200 with its receptor CD200R (marked with crossed out red lines) ❹, because the CD200/CD200R signaling provides immunosuppression due to the inhibition of macrophages, the induction of regulatory T cells, the switching of cytokine profiles from T helper-1 (Th1) to T helper-2 (Th2), the inhibition of tumor-specific T cell immunity, and the induction of myeloid-derived suppressor cells (MDSCs) [<a href="#B149-ijms-25-07527" class="html-bibr">149</a>]. Neuronal hyperactivity in diabetic encephalopathy may be caused by both neurotoxic factors outside microglia and an inflammatory response within microglia. Neurotoxic agents increase the risk of cognitive deficits due to impaired synaptic plasticity. Whatever the cause, neuronal hyperactivity has a feedback-activating effect on microglia by releasing slow-acting microglial activators, such as matrix metalloproteinase-9 (MMP-9), adenosine triphosphate (ATP), monocyte chemoattractant protein-1 (MCP-1), and fractalkine (CX3CL1) ❺. The soluble form of CX3CL1 (sCX3CL1) has a pro-inflammatory effect by stimulating metabotropic CX3CR1 receptors expressed in microglial cells ❻.</p>
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16 pages, 2522 KiB  
Article
KD-409, a Respiratory Syncytial Virus FG Chimeric Protein without the CX3C Chemokine Motif, Is an Efficient Respiratory Syncytial Virus Vaccine Preparation for Passive and Active Immunization in Mice
by Ryo Yamaue, Masaharu Torikai, Madoka Terashima and Hiroaki Mori
Vaccines 2024, 12(7), 753; https://doi.org/10.3390/vaccines12070753 - 8 Jul 2024
Viewed by 809
Abstract
Although respiratory syncytial virus (RSV) vaccine development initiatives have existed for half a century, no candidate has been approved for application at all ages from neonates to children. Developing an effective and safe RSV vaccine for pediatric use is challenging owing to RSV-associated [...] Read more.
Although respiratory syncytial virus (RSV) vaccine development initiatives have existed for half a century, no candidate has been approved for application at all ages from neonates to children. Developing an effective and safe RSV vaccine for pediatric use is challenging owing to RSV-associated disease and vaccine-enhanced disease (VED). We aimed to design an RSV vaccine, KD-409, by structurally incorporating the F ectodomain and G protein central conserved domain without the CX3C chemokine motif and test its efficacy and safety. KD-409 formed rosette particles or trimmers. KD-409 immunization of mice mainly induced anti-RSV F protein IgG. The induced anti-F antibodies had a higher IgG2a/IgG1 ratio than pre-fusion F, suggesting that they induced Th1-dominant immunity. Active and passive immunities were assessed by analyzing the viral titers in BALB/c mice intranasally challenged with RSV after intramuscular KD-409 immunization and pups derived from mothers who were intramuscularly vaccinated with KD-409 twice, respectively. KD-409 was more effective than post-fusion F and had a lower minimum effective dose than pre-fusion F. Thus, KD-409 demonstrated great potential as a novel RSV vaccine candidate, outperforming existing RSV F-based candidates. Our findings provide a promising strategy to overcome RSV-associated acute lower respiratory infections without the risk of VED associated with traditional approaches. Full article
(This article belongs to the Special Issue Novel Vaccines for Infectious Pathogens)
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<p>Design of respiratory syncytial virus (RSV) FG chimeric proteins. (<b>a</b>) KD-409 is based on the basic structure of RSV F sequence 1–513, except for the transmembrane region, and a portion of the FP domain, RSV F<sub>137–146</sub>, is replaced by RSV G<sub>162–171</sub>. Glycosylation is introduced around site IV, indicated by *. The K419N, K421T, T434N, and S436T mutations introduce N-type or O-type glycosylation. (<b>b</b>) Size-exclusion chromatography trace of trimeric and rosette-like particle structures. Positions of molecular weight standards are indicated with arrows. (<b>c</b>) Evaluation of the size distribution of KD-409 with dynamic light scattering. (<b>d</b>) Evaluation of KD-409, post-fusion F (post-F), and pre-fusion F (pre-F) using transmission electron microscopy. Inserts show zoomed views of each original image. Scale bar, 20 nm (pre-F) and 50 nm (post-F and KD-409).</p>
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<p>Design of respiratory syncytial virus (RSV) FG chimeric proteins. (<b>a</b>) KD-409 is based on the basic structure of RSV F sequence 1–513, except for the transmembrane region, and a portion of the FP domain, RSV F<sub>137–146</sub>, is replaced by RSV G<sub>162–171</sub>. Glycosylation is introduced around site IV, indicated by *. The K419N, K421T, T434N, and S436T mutations introduce N-type or O-type glycosylation. (<b>b</b>) Size-exclusion chromatography trace of trimeric and rosette-like particle structures. Positions of molecular weight standards are indicated with arrows. (<b>c</b>) Evaluation of the size distribution of KD-409 with dynamic light scattering. (<b>d</b>) Evaluation of KD-409, post-fusion F (post-F), and pre-fusion F (pre-F) using transmission electron microscopy. Inserts show zoomed views of each original image. Scale bar, 20 nm (pre-F) and 50 nm (post-F and KD-409).</p>
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<p>Evaluation of antibodies induced by KD-409. (<b>a</b>) Neutralizing antibody titers were measured in the absence of complement. The half-maximal inhibitory concentration (IC<sub>50</sub>) of pre-F and KD-409 immune sera are shown. Error bars indicate the 95% confidence interval (CI). Statistical analysis was performed with the two-tailed Mann–Whitney U test (** <span class="html-italic">p</span> &lt; 0.01, n = 8). (<b>b</b>) Neutralizing antibody titers were measured in the presence of complement. The IC<sub>50</sub> of pre-F and KD-409 immune sera are shown. Error bars indicate 95% CI. Statistical analysis was performed with the two-tailed Mann–Whitney U test (* <span class="html-italic">p</span> &lt; 0.05, n = 5–8). (<b>c</b>) IgG2a/IgG1 ratio of anti-F antibodies in immune sera is shown. Error bars indicate S.D. Statistical analysis was performed with the two-tailed Mann–Whitney U test (* <span class="html-italic">p</span> &lt; 0.05, pooled serum from 30 animals).</p>
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<p>Evaluation of protection against infection by active immunization. Mice inoculated twice with each protein antigen and phosphate–alum adjuvant (none) through intramuscular administration were used to evaluate protection against infection. RSV A2 at 1.0 × 10<sup>5</sup> PFU was inoculated intranasally, and the number of virus copies in bronchoalveolar lavage fluid (BALF) was measured 3 days after the virus challenge. Error bars indicate the 95% CI. (<b>a</b>) Infection-protective efficacy of pre-F, wild-type (WT) F, mutant F with a glycosylated modification of WT at site Ⅳ, and KD-409 (5 µg/dose antigen without Adju-Phos, n = 16). Statistical analysis was performed with the Kruskal–Wallis test and Dunn’s multiple comparison test (** <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). (<b>b</b>) Comparison of infection-protective efficacy between post-F and KD-409 (15 µg/dose antigen without Adju-Phos, n = 8). Statistical analysis was performed with the two-tailed Mann–Whitney U test (** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001). (<b>c</b>) Comparison of infection-protective efficacy between formalin-inactivated RSV (FI-RSV) and KD-409 (5 µg/dose antigen with Adju-Phos, n = 7–8). Statistical analysis is the same as in (<b>b</b>). LOD, limit of detection.</p>
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<p>Evaluation of protection against infection by active immunization. Mice inoculated twice with each protein antigen and phosphate–alum adjuvant (none) through intramuscular administration were used to evaluate protection against infection. RSV A2 at 1.0 × 10<sup>5</sup> PFU was inoculated intranasally, and the number of virus copies in bronchoalveolar lavage fluid (BALF) was measured 3 days after the virus challenge. Error bars indicate the 95% CI. (<b>a</b>) Infection-protective efficacy of pre-F, wild-type (WT) F, mutant F with a glycosylated modification of WT at site Ⅳ, and KD-409 (5 µg/dose antigen without Adju-Phos, n = 16). Statistical analysis was performed with the Kruskal–Wallis test and Dunn’s multiple comparison test (** <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). (<b>b</b>) Comparison of infection-protective efficacy between post-F and KD-409 (15 µg/dose antigen without Adju-Phos, n = 8). Statistical analysis was performed with the two-tailed Mann–Whitney U test (** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001). (<b>c</b>) Comparison of infection-protective efficacy between formalin-inactivated RSV (FI-RSV) and KD-409 (5 µg/dose antigen with Adju-Phos, n = 7–8). Statistical analysis is the same as in (<b>b</b>). LOD, limit of detection.</p>
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<p>Evaluation of protection against infection through passive immunization. Evaluation of protection against infection using pups born from mothers inoculated twice with each protein and phosphate–alum adjuvant through intramuscular administration. Pups were inoculated intranasally (i.n.) with RSV (A2) at 1.0 × 10<sup>5</sup> PFU. Virus copy numbers in lung tissue were measured 3 days after the virus challenge. (<b>a</b>) Overview of the animal study design. (<b>b</b>) Lung viral loads/virus copy numbers were measured using quantitative polymerase chain reaction (qPCR) (15 µg/dose antigen with Adju-Phos, n = 16). (<b>c</b>) Lung viral loads/virus copy numbers were measured using qPCR (0.5 ng/dose antigen with Adju-Phos, n = 16). Statistical analysis was performed with the Kruskal–Wallis test and Dunn’s multiple comparison test (<sup>ns</sup> <span class="html-italic">p</span> ≥ 0.05, ** <span class="html-italic">p</span> &lt; 0.01). Error bars are the 95% CI. i.m., intramuscular; LOD, limit of detection.</p>
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