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16 pages, 6164 KiB  
Article
Conserved Plastid Genomes of Pourthiaea Trees: Comparative Analyses and Phylogenetic Relationship
by Ting Ren, Chang Peng, Yuan Lu, Yun Jia and Bin Li
Forests 2024, 15(10), 1811; https://doi.org/10.3390/f15101811 - 16 Oct 2024
Viewed by 202
Abstract
The genus Pourthiaea Decne., a deciduous woody group with high ornamental value, belongs to the family Rosaceae. Here, we reported newly sequenced plastid genome sequences of Pourthiaea beauverdiana (C. K. Schneid.) Hatus., Pourthiaea parvifolia E. Pritz., Pourthiaea villosa (Thunb.) Decne., and Photinia glomerata [...] Read more.
The genus Pourthiaea Decne., a deciduous woody group with high ornamental value, belongs to the family Rosaceae. Here, we reported newly sequenced plastid genome sequences of Pourthiaea beauverdiana (C. K. Schneid.) Hatus., Pourthiaea parvifolia E. Pritz., Pourthiaea villosa (Thunb.) Decne., and Photinia glomerata Rehder & E. H. Wilson. The plastomes of these three Pourthiaea species shared the typical quadripartite structures, ranging in size from 159,903 bp (P. parvifolia) to 160,090 bp (P. beauverdiana). The three Pourthiaea plastomes contained a pair of inverted repeat regions (26,394–26,399 bp), separated by a small single-copy region (19,304–19,322 bp) and a large single-copy region (87,811–87,973 bp). A total of 113 unique genes were predicted for the three Pourthiaea plastomes, including four ribosomal RNA genes, 30 transfer RNA genes, and 79 protein-coding genes. Analyses of inverted repeat/single-copy boundary, mVISTA, nucleotide diversity, and genetic distance showed that the plastomes of 13 Pourthiaea species (including 10 published plastomes) are highly conserved. The number of simple sequence repeats and long repeat sequences is similar among 13 Pourthiaea species. The three non-coding regions (trnT-GGU-psbD, trnR-UCU-atpA, and trnH-GUG-psbA) were the most divergent. Only one plastid protein-coding gene, rbcL, was under positive selection. Phylogenetic analyses based on 78 shared plastid protein-coding sequences and 29 nrDNA sequences strongly supported the monophyly of Pourthiaea. As for the relationship with other genera in our phylogenies, Pourthiaea was sister to Malus in plastome phylogenies, while it was sister to the remaining genera in nrDNA phylogenies. Furthermore, significant cytonuclear discordance likely stems from hybridization events within Pourthiaea, reflecting complex evolutionary dynamics within the genus. Our study provides valuable genetic insights for further phylogenetic, taxonomic, and species delimitation studies in Pourthiaea, as well as essential support for horticultural improvement and conservation of the germplasm resources. Full article
(This article belongs to the Special Issue Biodiversity in Forests: Management, Monitoring for Conservation)
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<p>Plastome map of three <span class="html-italic">Pourthiaea</span> species. Genes located inside the circle are transcribed counterclockwise, and genes outside the circle are transcribed clockwise. The colored bars represent different functional groups of genes.</p>
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<p>Comparison of IR boundaries among 13 <span class="html-italic">Pourthiaea</span> plastomes.</p>
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<p>Simple sequence repeats (SSRs) in the 13 <span class="html-italic">Pourthiaea</span> plastomes. (<b>A</b>) Number of SSRs. (<b>B</b>) Number of SSR motifs.</p>
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<p>Long repeats in the 13 <span class="html-italic">Pourthiaea</span> plastomes. (<b>A</b>) Number of different repeat types. (<b>B</b>) Number of different repeat lengths. Forward (F), palindromic (P), reverse (R), and complementary (C) repeats.</p>
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<p>Comparison of the <span class="html-italic">Pourthiaea</span> plastome sequences using mVISTA with <span class="html-italic">P. villosa</span> as the reference.</p>
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<p>Nucleotide diversity (Pi) in whole plastomes. Coding (<b>A</b>) and non-coding (<b>B</b>) regions.</p>
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<p>The phylogenetic tree generated by BI and ML analyses based on 78 shared plastid protein-coding sequences and 29 nrDNA sequences with <span class="html-italic">Photinia glomerata</span> as outgroup. ML replicate values and BI posterior probability values are given at each node. “*” represents the highest support (100%/1). “#” represents that the node does not occur in the ML tree.</p>
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11 pages, 1043 KiB  
Article
Effect of Food Additive E171 and Titanium Dioxide Nanoparticles (TiO2-NPs) on Caco-2 Colon Cancer Cells
by Ewa Baranowska-Wójcik, Anna Rymuszka, Anna Sierosławska and Dominik Szwajgier
Appl. Sci. 2024, 14(20), 9387; https://doi.org/10.3390/app14209387 - 15 Oct 2024
Viewed by 279
Abstract
The food coloring agent E171 raises many questions concerning its negative impact on human health because of the fact that it contains nanoparticle fractions (NPs, diameter < 100 nm). Numerous studies showed its influence on organisms, including the ability to disrupt the intestinal [...] Read more.
The food coloring agent E171 raises many questions concerning its negative impact on human health because of the fact that it contains nanoparticle fractions (NPs, diameter < 100 nm). Numerous studies showed its influence on organisms, including the ability to disrupt the intestinal barrier. In the present study, we verified the potential toxicity and pro-inflammatory activity of three different E171 samples (containing NPs fractions) and one TiO2 NPs sample (60–600 µg mL−1) towards Caco-2 colon cancer cells. The experiments revealed no significant changes in terms of the vitality of Caco-2 cells after 24 h of exposure (XTT test). However, after 72 h, a decrease in the proliferation of Caco-2 cells caused by three TiO2 substances was observed. Moreover, deterioration of the metabolic activity of Caco-2 cells (ATP test) by all analyzed substances at 300 and 600 µg mL−1 was seen. While a 24-h exposure to each tested substance resulted in a negligible release of LDH, a prolonged exposure (72 h) indicated an elevated release of LDH, suggesting potential toxicity. All TiO2 samples induced the elevated release of two primary proinflammatory cytokines, i.e., IL-1β and TNF-α, in a dose-independent manner. The discrepancies in the results come from the differences in the share of individual sizes in four TiO2 products. Full article
(This article belongs to the Section Food Science and Technology)
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<p>Effect of the tested substances (S1–S4) on the proliferation of Caco-2 cells after 24-h (<b>A</b>) and 72-h (<b>B</b>) exposures measured with the XTT assay. Ctr neg—negative control, Ctr pos—positive control (mean ± SD, * significantly different from the negative control at <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Effect of the tested substances (S1–S4) on the metabolic activity of Caco-2 cells after 24 h (<b>A</b>) and 72 h (<b>B</b>) exposures assessed by measuring cellular ATP content. Values are expressed as mean ± SE. The asterisk represents a statistical difference at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Effect of the tested substances (S1–S4) on the cell membrane integrity of Caco-2 cells after 24-h (<b>A</b>) and 72-h (<b>B</b>) exposures assessed by measuring the release of lactate dehydrogenase (LDH) from cells. Values are expressed as mean ± SE. The asterisk represents a statistical difference at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Effect of the tested substances (S1–S4) on the level of pro-inflammatory IL-1ß (<b>A</b>) and TNF-α cytokine (<b>B</b>) in Caco-2 cells after a 72 h exposure, measured with the ELISA assay. Ctr pos—positive control, was defined as LPS-treated cells at 30 µg mL<sup>−1</sup> (mean ± SD, * significantly different from the control (Ctr) at <span class="html-italic">p</span> &lt; 0.05).</p>
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16 pages, 3838 KiB  
Article
Identification of Listeria Isolates by Using a Pragmatic Multilocus Phylogenetic Analysis
by Antonio Martínez-Murcia, Aaron Navarro and Caridad Miró-Pina
Microbiol. Res. 2024, 15(4), 2114-2128; https://doi.org/10.3390/microbiolres15040142 (registering DOI) - 14 Oct 2024
Viewed by 214
Abstract
Species identification of Listeria isolates remained a tedious process still based on culturing methods that, in recent years, have led to the description of many species that are not even part of the genus Listeria. It is advisable to provide new precise [...] Read more.
Species identification of Listeria isolates remained a tedious process still based on culturing methods that, in recent years, have led to the description of many species that are not even part of the genus Listeria. It is advisable to provide new precise techniques since this taxon includes two pathogens that are usually transmitted through the food chain, Listeria monocytogenes and L. ivanovii. The approach, so-called multilocus phylogenetic analysis (MLPA) that uses several concatenated housekeeping gene sequences, provides accurate and affordable classification frameworks to easily identify Listeria species by simple Sanger sequencing. Fragments of seven housekeeping genes (gyrA, cpn60, parE, recA, rpoB, atpA, and gyrB) from 218 strains of all Listeria species currently described were used to build an MLPA of the concatenated sequence, a total of 4375 bp. All isolates subjected to identification were clustered within the species of Listeria sensu stricto, L. monocytogenes, L. innocua, and L. welshimeri, and some reference strains were reclassified as L. ivanovii and L. seeligeri. Housekeeping-gene sequencing has been demonstrated to represent a pragmatic tool that can be firmly considered in food control. Full article
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<p>Neighbor-joining phylogenetic tree based on the analysis of 16S rRNA gene sequences (935 bp) of all described <span class="html-italic">Listeria</span> sensu stricto and <span class="html-italic">Listeria</span> sensu lato species, routed using <span class="html-italic">Bacillus cereus</span>. Numbers at nodes indicate bootstrap values (percentage of 1000 replicates). <sup>T</sup>—type strains.</p>
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<p>Neighbor-joining phylogenetic tree based on the MLPA from seven concatenated housekeeping genes (<span class="html-italic">gyrA</span>, <span class="html-italic">cpn60</span>, <span class="html-italic">parE</span>, <span class="html-italic">recA</span>, <span class="html-italic">rpoB</span>, <span class="html-italic">atpA</span> and <span class="html-italic">gyrB</span>; a total of 4375 bp) of strains of all described <span class="html-italic">Listeria</span> sensu stricto and sensu lato species, <span class="html-italic">Bacillus cereus</span>, <span class="html-italic">Brochothrix thermosphacta</span>, and <span class="html-italic">Streptococcus pneumoniae</span>. Numbers at nodes indicate bootstrap values (percentage of 1000 replicates). Strains sequenced in this study are shown in bold. <sup>T</sup>—type strains.</p>
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<p>Graphical representation of the ranges of intra- and inter-species nucleotide substitution percentages in red and blue bars, respectively, and intra- and inter-species phylogenetic depth (black) for the concatenated seven-gene sequence, calculated for all <span class="html-italic">Listeria</span> sensu stricto species and subspecies and <span class="html-italic">L. monocytogenes</span> genetic lineages.</p>
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<p>Neighbor-joining phylogenetic tree based on the MLPA from seven concatenated housekeeping genes (<span class="html-italic">gyrA</span>, <span class="html-italic">cpn60</span>, <span class="html-italic">parE</span>, <span class="html-italic">recA</span>, <span class="html-italic">rpoB</span>, <span class="html-italic">atpA</span>, and <span class="html-italic">gyrB</span>; a total of 4375 bp) of strains of all described <span class="html-italic">Listeria</span> sensu stricto, including non-characterized isolates. Numbers at nodes indicate bootstrap values (percentage of 1000 replicates). Strains sequenced in this study are shown in bold and <span class="html-italic">Listeria</span> isolates identified in this study are shown in red. <sup>T</sup>—type strains.</p>
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<p>Neighbor-joining phylogenetic tree based on the MLPA from seven concatenated housekeeping genes (<span class="html-italic">gyrA</span>, <span class="html-italic">cpn60</span>, <span class="html-italic">parE</span>, <span class="html-italic">recA</span>, <span class="html-italic">rpoB</span>, <span class="html-italic">atpA</span>, and <span class="html-italic">gyrB</span>; a total of 4375 bp) of strains from the four <span class="html-italic">L. monocytogenes</span> genetic lineages. Numbers at nodes indicate bootstrap values (percentage of 1000 replicates). Strains sequenced in this study are shown in bold and <span class="html-italic">Listeria</span> isolates identified in this study are shown in red. <sup>T</sup>—type strains.</p>
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28 pages, 2779 KiB  
Review
Anaerobic Sport-Specific Tests for Taekwondo: A Narrative Review with Guidelines for the Assessment
by Gennaro Apollaro, Ibrahim Ouergui, Yarisel Quiñones Rodríguez, Rafael L. Kons, Daniele Detanico, Emerson Franchini, Piero Ruggeri, Coral Falcó and Emanuela Faelli
Sports 2024, 12(10), 278; https://doi.org/10.3390/sports12100278 - 14 Oct 2024
Viewed by 609
Abstract
The ATP-PCr system represents the main source of energy during high-intensity attack actions in taekwondo matches. In contrast, the glycolytic system supports the maintenance of these actions when repeated techniques are performed. Given the close relationship between anaerobic energy systems and attack activity [...] Read more.
The ATP-PCr system represents the main source of energy during high-intensity attack actions in taekwondo matches. In contrast, the glycolytic system supports the maintenance of these actions when repeated techniques are performed. Given the close relationship between anaerobic energy systems and attack activity in combat, the literature relating to the use of sport-specific test protocols for anaerobic assessment has experienced a remarkable increase. This narrative review aims to illustrate the sport-specific anaerobic tests available in taekwondo by retracing and examining development and validation process for each test. Forty-one articles published between 2014 and 2023 were selected via the MEDLINE and Google Scholar bibliographic databases. These tests are the Taekwondo Anaerobic Test and Adapted Anaerobic Kick Test (i.e., continuous mode testing); the 10 s and multiple Frequency Speed of Kick Tests; the chest and head Taekwondo Anaerobic Intermittent Kick Tests; and the Taekwondo-Specific Aerobic–Anaerobic–Agility test (i.e., intermittent mode testing). Coaches and strength and conditioning professionals can use all the tests described in taekwondo gyms as they feature short and easy-to-implement protocols for monitoring and prescribing specific anaerobic training. The guidelines in this review evaluate each test from several perspectives: basic (e.g., validity, reliability, and sensitivity), methodological (e.g., continuous or intermittent mode testing) and application (e.g., time–motion structure and performance parameters). This comprehensive approach aims to assist stakeholders in selecting the most appropriate test. Full article
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<p><b>Blood lactate values [La] post-rounds in simulated and official match</b>. First, some studies have quantified the [La] after each round of the match. Specific analysis of these values reveals a gradual increase in the values of [La] post-round, from round to round, with the highest value at the end of the last round of the match, in both simulated and official matches. However, data should be interpreted with caution as this increase throughout the rounds does not necessarily reflect glycolytic contribution increase [<a href="#B25-sports-12-00278" class="html-bibr">25</a>]. Indeed, the studies [<a href="#B17-sports-12-00278" class="html-bibr">17</a>,<a href="#B18-sports-12-00278" class="html-bibr">18</a>,<a href="#B19-sports-12-00278" class="html-bibr">19</a>,<a href="#B24-sports-12-00278" class="html-bibr">24</a>] that also calculated delta (Δ) [La] (i.e., the lactate concentration after the round minus the lactate concentration at the beginning of the round) suggest a decrease in lactate accumulation and a consequent reduction in glycolytic participation throughout the rounds. Moreover, it is important to note that identifying [La]<sub>peak</sub> after each round is not practicable after rounds 1 and 2 due to the short duration of recovery (i.e., 1 min) between rounds. Secondly, placing the [La] values in ascending order, for each round, shows that the values identified in simulated matches, in which the contribution of the three energy systems was quantified in parallel, are slightly lower (and with a certain degree of overlap for the values after the third round) than those found in simulated and official matches, in which only the contribution of the glycolytic system was quantified or a backpack was used to protect the portable gas analysis system. Values: mean ± SD.</p>
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<p><b>Anaerobic continuous sport-specific tests. Taekwondo Anaerobic Test (TAT)</b> [<a href="#B38-sports-12-00278" class="html-bibr">38</a>,<a href="#B51-sports-12-00278" class="html-bibr">51</a>]. Athlete performs the bandal-chagi by alternating the legs, beginning with dominant leg, as many times as possible at maximal intensity over 30 s. Kicks must be carried out in height between the umbilical scar and xiphoid process of the athlete, marked by placing a taekwondo body protector on the punching bag. The kicking cycle is defined as the time interval between two consecutive kicks with the same leg. From this parameter, the number of kicking cycles (only completed cycles), mean kicking time and best kicking time are calculated. In addition, by measuring the magnitude of impact in each kick, the highest kicking impact and the mean kicking impact over the 30 s of the test are identified. The fatigue index (FI) is calculated using the mean kicking time and mean impact of the initial 20% cycles and the mean of the last 20% cycles [<a href="#B38-sports-12-00278" class="html-bibr">38</a>]. During the test, the amount of performed techniques are recorded, as well as the kicking impact force. Consequently, the following performance indicators are calculated: Peak power observed during the first 5 s of the test; relative peak power; mean anaerobic power during the 30 s of the test; relative mean anaerobic power; fatigue index; and anaerobic capacity [<a href="#B51-sports-12-00278" class="html-bibr">51</a>]. <b>Adapted Anaerobic Kick Test (AAKT)</b> [<a href="#B48-sports-12-00278" class="html-bibr">48</a>]. Athlete performs the bandal-chagi with the dominant leg as many times as possible at maximal intensity over 30 s. The test is performed using a target pad positioned at the height of the iliac crest of the athlete. The first kick is performed with the dominant leg in the back. Starting from the second kick, the preferred leg is positioned forward throughout the remaining time of the test. Only kicks performed with the front leg are considered for the analysis. The following parameters are calculated: higher kick frequency performed during 3 s; lower kick frequency performed during 3 s; average kick frequency performed during 30 s; fatigue index (percentage reduction of the maximum frequency kick to minimum frequency kick); and time to higher kick frequency performed during 3 s.</p>
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<p><b>Anaerobic intermittent sport-specific tests.</b> (<b>a</b>) <b>10 s Frequency Speed of Kick Test (FSKT<sub>10s</sub>)</b> [<a href="#B56-sports-12-00278" class="html-bibr">56</a>]. The FSKT<sub>10s</sub> lasts for 10 s. After the sound signal, athlete must execute the maximum number of bandal-chagi movements possible by alternating right and left legs. In order to accomplish the test, each athlete is placed in front of the stand bag equipped with a taekwondo body protector, positioned at the same height of the athlete trunk. The performance is determined by the total number of kicks applied during the test. (<b>b</b>) <b>Multiple Frequency Speed of Kick Test (FSKT<sub>mult</sub>)</b> [<a href="#B56-sports-12-00278" class="html-bibr">56</a>]. The FSKT<sub>mult</sub> consists of five 10 s sets with a 10 s passive recovery between sets. The execution criteria for the FSKT<sub>mult</sub> are the same as those defined for the FSKT<sub>10s</sub>. The performance is determined by the number of kicks in each set, total number of kicks and kick decrement index (KDI) during the test. The KDI indicates performance decreases during the test. To calculate the KDI, the following Equation is used, which takes into account the number of kicks applied during all sets of the FSKT<sub>mult</sub>: KDI (%) = [1 − (FSKT<sub>1</sub> + FSKT<sub>2</sub> + FSKT<sub>3</sub> + FSKT<sub>4</sub> + FSKT<sub>5</sub>)/best FSKT × number of sets] × 100. <b>Video Analysis</b>. Both tests are recorded, and the videos are analyzed posteriorly to manually count the kicks performed, through video analysis software. First, the count starts when the athlete moves the attack feet and finishes when he touches the bag. Valid kicks are those that hit the target during 10 s. If the athlete starts the kick before completing 10 s but reaches the target only after 10 s, the kick is not considered valid. Second, the valid kicks are those performed with appropriate technique and power.</p>
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<p><b>Anaerobic intermittent sport-specific tests. Taekwondo Anaerobic Intermittent Kick Test (TAIKT<sub>chest</sub>)</b> [<a href="#B27-sports-12-00278" class="html-bibr">27</a>]. The TAIKT<sub>chest</sub> consists of six 5 s sets with a 10 s active recovery (i.e., very light [tempo = one bounce/s] bouncing movements controlled by an evaluator) between sets. After the sound signal, athlete must execute the maximum number of bandal-chagi movements possible by alternating right and left legs. In order to accomplish the test, each athlete is placed in front of the stand bag equipped with a taekwondo electronic body protector, positioned at the same height of the athlete trunk, i.e., at a height (y) relative to the mat. During kick execution, the athlete should not exceed a mark on the mat, the optimum distance (x) to be determined before the test, to effectively execute kicking on the body protector. The distances (x) and (y) allow to determine the distance (d) using the Pythagorean Theorem, which is the projection distance of the foot on the body protector. Participants are asked to wear their official protectors during the test. The number of kicks is automatically displayed on the computer screen after each kicking set and the scoring threshold is set according to the criteria used in the competition for each weight category. TAIKT<sub>chest</sub> performance is expressed as absolute (W) and relative (W·kg<sup>−0.67</sup>) peak power (P<sub>peakTAIKT</sub>) and mean power (P<sub>meanTAIKT</sub>), and absolute (W) fatigue index (FI<sub>TAIKT</sub>). P<sub>peakTAIKT</sub> is the highest power output of the six sets of kicks; P<sub>meanTAIKT</sub> is sum of powers of six sets of kicks/6; FI<sub>TAIKT</sub> is P<sub>peakTAIKT</sub>-minimum power (P<sub>minTAIKT</sub>)/total test duration (30 s). The authors have made an Excel spreadsheet available in which performance can be calculated by entering the known values, i.e., body mass, x and y distances, and number of kicks in each series. <b>Taekwondo Anaerobic Intermittent Kick Test (TAIKT<sub>head</sub>)</b> [<a href="#B57-sports-12-00278" class="html-bibr">57</a>]. The execution criteria and performance for the TAIKT<sub>head</sub> are the same as those defined for the TAIKT<sub>chest</sub>, except that in the TAIKT<sub>head</sub>, the kicks are projected on a dummy’s head covered by an electronic head protector.</p>
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<p><b>Anaerobic intermittent sport-specific test. Taekwondo-Specific Aerobic–Anaerobic–Agility (TAAA) test</b> [<a href="#B39-sports-12-00278" class="html-bibr">39</a>]. The test involves six 20 s (a total of 2 min) intervals of shuttle sprints over a 4 m distance, and the execution of the bandal-chagi to the punching bags alternating the legs at the end of each distance, with 10 s rest intervals between the sets. For a detailed description of this test, refer to the previous review on sport-specific assessment of endurance in taekwondo [<a href="#B9-sports-12-00278" class="html-bibr">9</a>]. To estimate anaerobic fitness are calculated: maximum kicks (maximum number of kicks in a 20 s interval); minimum kicks (minimum number of kicks in a 20 s interval); average kicks (total number of kicks at the end of the test divided by six) and kick fatigue index (KFI) according to following Equation: KFI (%) = [Maximum Kicks − Minimum Kicks)/Total Kicks] × 100.</p>
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12 pages, 985 KiB  
Article
Alternative DNA Markers to Detect Guam-Specific CRB-G (Clade I) Oryctes rhinoceros (Coleoptera: Scarabaeidae) Indicate That the Beetle Did Not Disperse from Guam to the Solomon Islands or Palau
by Wee Tek Tay, Sean D. G. Marshall, Angel David Popa-Baez, Glenn F. J. Dulla, Andrea L. Blas, Juniaty W. Sambiran, Meldy Hosang, Justine Bennette H. Millado, Michael Melzer, Rahul V. Rane, Tim Hogarty, Demi Yi-Chun Cho, Jelfina C. Alouw, Muhammad Faheem and Benjamin D. Hoffmann
Diversity 2024, 16(10), 634; https://doi.org/10.3390/d16100634 - 10 Oct 2024
Viewed by 592
Abstract
A partial mitochondrial DNA Cytochrome Oxidase subunit I (mtCOI) gene haplotype variant of the coconut rhinoceros beetle (CRB) Oryctes rhinoceros, classed as ‘CRB-G (clade I)’, has been the focus of much research since 2007, with reports of invasions into new [...] Read more.
A partial mitochondrial DNA Cytochrome Oxidase subunit I (mtCOI) gene haplotype variant of the coconut rhinoceros beetle (CRB) Oryctes rhinoceros, classed as ‘CRB-G (clade I)’, has been the focus of much research since 2007, with reports of invasions into new Pacific Island locations (e.g., Guam, Hawaii, Solomons Islands). For numerous invasive species, inference of invasion biology via whole genome is superior to assessments via the partial mtCOI gene. Here, we explore CRB draft mitochondrial genomes (mitogenomes) from historical and recent collections, with assessment focused on individuals associated within the CRB-G (clade I) classification. We found that all Guam CRB individuals possessed the same mitogenome across all 13 protein-coding genes and differed from individuals collected elsewhere, including ‘non-Guam’ individuals designated as CRB-G (clade I) by partial mtCOI assessment. Two alternative ATP6 and COIII partial gene primer sets were developed to enable distinction between CRB individuals from Guam that classed within the CRB-G (clade I) haplotype grouping and CRB-G (Clade I) individuals collected elsewhere. Phylogenetic analyses based on concatenated ATP6–COIII genes showed that only Guam CRB-G (clade I) individuals clustered together, and therefore Guam was not the source of the CRB that invaded the other locations in the Pacific assessed in this study. The use of the mtCOI and/or mtCOIII genes for initial molecular diagnosis of CRB remained crucial, and assessment of more native CRB populations will further advance our ability to identify the provenance of CRB invasions being reported within the Pacific and elsewhere. Full article
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<p>Phylogenetic analysis using (<b>a</b>) partial mt<span class="html-italic">COI</span> gene sequence (676 bp) and (<b>b</b>) concatenated partial <span class="html-italic">APT6</span> (446 bp) and partial <span class="html-italic">COIII</span> (422 bp) gene sequences. A phylogram based on concatenated <span class="html-italic">ATP6–COIII</span> partial gene sequences and the haplotype network are also presented in (<b>c</b>) and (<b>d</b>), respectively. The <span class="html-italic">Oryctes narsicornis</span> sample (OK484312) was included as an outgroup.</p>
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17 pages, 3897 KiB  
Article
Blue Mussel-Derived Bioactive Peptides PIISVYWK (P1) and FSVVPSPK (P2): Promising Agents for Inhibiting Foam Cell Formation and Inflammation in Cardiovascular Diseases
by Chathuri Kaushalya Marasinghe and Jae-Young Je
Mar. Drugs 2024, 22(10), 466; https://doi.org/10.3390/md22100466 - 10 Oct 2024
Viewed by 427
Abstract
Atherosclerosis is a key etiological event in the development of cardiovascular diseases (CVDs), strongly linked to the formation of foam cells. This study explored the effects of two blue mussel-derived bioactive peptides (BAPs), PIISVYWK (P1) and FSVVPSPK (P2), on inhibiting foam cell formation [...] Read more.
Atherosclerosis is a key etiological event in the development of cardiovascular diseases (CVDs), strongly linked to the formation of foam cells. This study explored the effects of two blue mussel-derived bioactive peptides (BAPs), PIISVYWK (P1) and FSVVPSPK (P2), on inhibiting foam cell formation and mitigating inflammation in oxLDL-treated RAW264.7 macrophages. Both peptides significantly suppressed intracellular lipid accumulation and cholesterol levels while promoting cholesterol efflux by downregulating cluster of differentiation 36 (CD36) and class A1 scavenger receptors (SR-A1) and upregulating ATP binding cassette subfamily A member 1 (ABCA-1) and ATP binding cassette subfamily G member 1 (ABCG-1) expressions. The increased expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α) further validated their role in enhancing cholesterol efflux. Additionally, P1 and P2 inhibited foam cell formation in oxLDL-treated human aortic smooth muscle cells and exerted anti-inflammatory effects by reducing pro-inflammatory cytokines, nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), primarily through inhibiting NF-κB activation. Furthermore, P1 and P2 alleviated oxidative stress by activating the Nrf2/HO-1 pathway. Our findings demonstrate that P1 and P2 have significant potential in reducing foam cell formation and inflammation, both critical factors in atherosclerosis development. These peptides may serve as promising therapeutic agents for the prevention and treatment of CVDs associated with oxidative stress and inflammation. Full article
(This article belongs to the Special Issue Marine Anti-inflammatory and Antioxidant Agents 4.0)
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<p>Chemical structures of PIISVYWK (P1) and FSVVPSPK (P2).</p>
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<p>(<b>A</b>) Cell viability, (<b>B</b>) inhibition of intracellular lipid accumulation quantitatively, and (<b>C</b>) qualitatively (40× magnification) of PIISVYWK (P1) and FSVVPSPK (P2) peptides in oxLDL-treated RAW264.7 macrophages. The macrophages were treated with P1 and P2 peptides or positive controls (10 µM) including simvastatin (SIM) or rosiglitazone (RSG) or oxLDL (50 µg/mL) for MTT assay. For ORO staining, macrophages were treated with P1 and P2 peptides or positive controls (10 µM) including SIM or RSG for 1 h followed by oxLDL treatment for another 24 h. Experiments consisted of three independent measurements (<span class="html-italic">n</span> = 3) with ± S.D. ** <span class="html-italic">p</span> &lt; 0.001 and <sup>##</sup> <span class="html-italic">p</span> &lt; 0.001, compared to the oxLDL-treated group and non-treated group, respectively. The numbers behind letters in images denote concentrations in µM.</p>
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<p>Effect of 10~200 µM of P1 and P2 peptides on (<b>A</b>) total cholesterol, (<b>B</b>) free cholesterol, (<b>C</b>) cholesterol ester, and (<b>D</b>) triglycerides content in oxLDL-treated RAW264.7 macrophages. The macrophages were treated with P1 and P2 peptides for 1 h followed by oxLDL treatment for 24 h. Experiments consisted of three independent measurements (<span class="html-italic">n</span> = 3) with ± S.D. ** <span class="html-italic">p</span> &lt; 0.001 and <sup>##</sup> <span class="html-italic">p</span> &lt; 0.001, compared to the oxLDL-treated group and non-treated group, respectively.</p>
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<p>Effect of 10~200 µM of P1 and P2 peptides on (<b>A</b>) cholesterol influx, (<b>B</b>) cholesterol efflux, (<b>C</b>) ABCA-1, ABCG-1, SR-A1, and CD36 protein expressions in oxLDL-treated RAW264.7 macrophages. The macrophages were treated with P1 and P2 peptides for 1 h followed by oxLDL treatment for 24 h. Experiments consisted of three independent measurements (<span class="html-italic">n</span> = 3) with ± S.D. ** <span class="html-italic">p</span> &lt; 0.001 and <sup>##</sup> <span class="html-italic">p</span> &lt; 0.001, compared to the oxLDL-treated group and non-treated group, respectively. <span class="html-italic">* p</span> &lt; 0.05 compared to the oxLDL-treated group.</p>
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<p>Effect of 10~200 µM of P1 and P2 peptides on (<b>A</b>) PPAR-γ and LXR-α protein expressions in oxLDL-treated RAW264.7 macrophages and (<b>B</b>) foam cell formation inhibition in hASMCs. The macrophages and hASMCs were treated with P1 and P2 peptides for 1 h followed by oxLDL treatment for 24 h. Relevant images are taken at 20× magnification. Experiments consisted of three independent measurements (<span class="html-italic">n</span> = 3) with ± S.D. ** <span class="html-italic">p</span> &lt; 0.001 and <sup>##</sup> <span class="html-italic">p</span> &lt; 0.001, compared to the oxLDL-treated group and non-treated group, respectively. * <span class="html-italic">p</span> &lt; 0.05 compared to the oxLDL-treated group. In images, numbers behind letters represent concentrations in µM.</p>
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<p>Effect of P1 and P2 peptides on pro-inflammatory cytokine production (<b>A</b>) TNF-α, (<b>B</b>) IL-6, and (<b>C</b>) IL-1β in oxLDL-treated RAW264.7 macrophages. The macrophages were treated with P1 and P2 peptides for 1 h followed by oxLDL treatment for 24 h. Experiments consisted of three independent measurements (<span class="html-italic">n</span> = 3) with ± S.D. ** <span class="html-italic">p</span> &lt; 0.001 and <sup>##</sup> <span class="html-italic">p</span> &lt; 0.001, compared to the oxLDL-treated group and non-treated group, respectively.</p>
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<p>Effect of P1 and P2 peptides on (<b>A</b>) NO production, (<b>B</b>) PGE<sub>2</sub> production, (<b>C</b>) iNOS, COX-2, and HO-1 protein expression in oxLDL-treated RAW264.7 macrophages. The macrophages were treated with P1 and P2 peptides for 1 h followed by oxLDL treatment for 24 h. Experiments consisted of three independent measurements (<span class="html-italic">n</span> = 3) with ± S.D. ** <span class="html-italic">p</span> &lt; 0.001 and <sup>##</sup> <span class="html-italic">p</span> &lt; 0.001, compared to the oxLDL-treated group and non-treated group, respectively. * <span class="html-italic">p</span> &lt; 0.05 compared to the oxLDL-treated group. (<b>D</b>) NF-κB nuclear activation and (<b>E</b>) Nrf2 nuclear translocation in oxLDL-treated RAW264.7 macrophages. The macrophages were treated with peptides for 1 h followed by oxLDL (50 µg/mL) treatment for 2 h for NF-κB and Nrf2 immunostaining. The numbers behind letters in images denote concentrations in µM.</p>
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<p>Schematic diagram for proposed mechanism.</p>
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18 pages, 2442 KiB  
Article
Cytotoxic Potencies of Zinc Oxide Nanoforms in A549 and J774 Cells
by Nazila Nazemof, Dalibor Breznan, Yasmine Dirieh, Erica Blais, Linda J. Johnston, Azam F. Tayabali, James Gomes and Premkumari Kumarathasan
Nanomaterials 2024, 14(19), 1601; https://doi.org/10.3390/nano14191601 - 3 Oct 2024
Viewed by 817
Abstract
Zinc oxide nanoparticles (NPs) are used in a wide range of consumer products and in biomedical applications, resulting in an increased production of these materials with potential for exposure, thus causing human health concerns. Although there are many reports on the size-related toxicity [...] Read more.
Zinc oxide nanoparticles (NPs) are used in a wide range of consumer products and in biomedical applications, resulting in an increased production of these materials with potential for exposure, thus causing human health concerns. Although there are many reports on the size-related toxicity of ZnO NPs, the toxicity of different nanoforms of this chemical, toxicity mechanisms, and potency determinants need clarification to support health risk characterization. A set of well-characterized ZnO nanoforms (e.g., uncoated ca. 30, 45, and 53 nm; coated with silicon oil, stearic acid, and (3-aminopropyl) triethoxysilane) were screened for in vitro cytotoxicity in two cell types, human lung epithelial cells (A549), and mouse monocyte/macrophage (J774) cells. ZnO (bulk) and ZnCl2 served as reference particles. Cytotoxicity was examined 24 h post-exposure by measuring CTB (viability), ATP (energy metabolism), and %LDH released (membrane integrity). Cellular oxidative stress (GSH-GSSG) and secreted proteins (targeted multiplex assay) were analyzed. Zinc oxide nanoform type-, dose-, and cell type-specific cytotoxic responses were seen, along with cellular oxidative stress. Cell-secreted protein profiles suggested ZnO NP exposure-related perturbations in signaling pathways relevant to inflammation/cell injury and corresponding biological processes, namely reactive oxygen species generation and apoptosis/necrosis, for some nanoforms, consistent with cellular oxidative stress and ATP status. The size, surface area, agglomeration state and metal contents of these ZnO nanoforms appeared to be physicochemical determinants of particle potencies. These findings warrant further research on high-content “OMICs” to validate and resolve toxicity pathways related to exposure to nanoforms to advance health risk-assessment efforts and to inform on safer materials. Full article
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<p>Cell morphology observed after exposure of A549 and J774 to the different doses of ZnO nanoparticles (e.g., (<b>A</b>) UC-2 and (<b>B</b>) AM): light microscopy images (40× magnification).</p>
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<p>Cytotoxicity in A549 cells (mean ± SEM) after exposure (24 h) to ZnO nanoforms and the reference particles. Exposure experiments were conducted three times (n = 3), with duplicate samples per treatment group in each exposure experiment. (<b>A</b>) LDH Release, (<b>B</b>) CTB (Resazurin) Reduction, (<b>C</b>) Cellular ATP Levels.</p>
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<p>Cytotoxicity in J774 cells (mean ± SEM) after exposure (24 h) to ZnO nanoforms and reference particles. Exposure experiments were conducted three times (n = 3), with duplicate samples per treatment group in each exposure experiment. (<b>A</b>) LDH Release, (<b>B</b>) CTB (Resazurin) Reduction, (<b>C</b>) Cellular ATP Levels.</p>
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<p>Cellular oxidative stress status in (<b>A</b>) A549 and (<b>B</b>) J774 cells after exposure to ZnO NPs, as well as to the reference particles (30 µg/cm<sup>2</sup>).</p>
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<p>Heatmap and hierarchical clustering of secreted protein fold changes normalized to control (24 h post exposure of cells to ZnO nanoforms and reference particles: (<b>A</b>) A549 and (<b>B</b>) J774). Red—increased; green—decreased.</p>
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<p>Pathway analysis results for in vitro cellular exposure (24 h) to ZnO nanoforms and the reference particles ((<b>A</b>) A549 and (<b>B</b>) J774). Orange—increased; blue—decreased.</p>
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16 pages, 14233 KiB  
Article
Sequential Immune Acquisition of Monoclonal Antibodies Enhances Phagocytosis of Acinetobacter baumannii by Recognizing ATP Synthase
by Dong Huang, Zhujun Zeng, Zhuolin Li, Mengjun Li, Linlin Zhai, Yuhao Lin, Rui Xu, Jiuxin Qu, Bao Zhang, Wei Zhao and Chenguang Shen
Vaccines 2024, 12(10), 1120; https://doi.org/10.3390/vaccines12101120 - 29 Sep 2024
Viewed by 521
Abstract
Objectives: The aim of this study was to prepare monoclonal antibodies (mAbs) that broadly target Acinetobacter baumannii and protect against infection by multi-drug-resistant (MDR) A. baumannii from different sources. Methods: mAb 8E6 and mAb 1B5 were prepared by sequentially immunizing mice [...] Read more.
Objectives: The aim of this study was to prepare monoclonal antibodies (mAbs) that broadly target Acinetobacter baumannii and protect against infection by multi-drug-resistant (MDR) A. baumannii from different sources. Methods: mAb 8E6 and mAb 1B5 were prepared by sequentially immunizing mice with a sublethal inoculation of three heterogeneous serotypes of pan-drug-resistant (PDR) A. baumannii, ST-208, ST-195, and ST-229. Results: The cross-recognition of heterogeneous bacteria (n = 13) by two mAbs and potential targets was verified, and the in vitro antibacterial efficacy of mAbs was assessed. The median killing rate of mAb 8E6 against A. baumannii in the presence of complement and dHL-60 cells was found to be 61.51%, while that of mAb 1B5 was 41.96%. When only dHL-60 cells were present, the killing rate of mAb 8E6 was 65.73%, while that of mAb 1B5 was 69.93%. We found that mAb 8E6 and mAb 1B5 broadly targeted MDR A. baumannii on the ATP synthase complex and were equipped with an antibacterial killing ability by enhancing the innate immune bacteriolytic effect of ST-208 and ST-195 strains. Both monoclonal antibodies were validated to protect against respiratory infection at 4 and 24 h via enhancing the release of innate immune substances and inflammatory cytokines, effectively shortening the disease period in mice. Conclusions: mAb 8E6 and mAb 1B5 significantly enhanced the opsonization process of phagocytosis against A. baumannii strains prevalent in southern China by targeting ATP synthase antigens thereof, resulting in protective effects in mice. Full article
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<p>Each round of the sequential immunization strategy utilizes one of the three strains of PDR <span class="html-italic">A. baumannii</span>, eliciting strong cross-immune responses from sera that are generated through multiple rounds of immunization. The data are presented as the mean ± standard deviation (SD). A two-way ANOVA was conducted; *, <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 versus isotype mAb group. There was no statistically significant difference in the OD450 nm values among the three strains of <span class="html-italic">A. baumannii</span> when comparing the two distinct continuous detection gradients.</p>
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<p>(<b>A</b>) The mAbs-mediated complement and neutrophil bactericidal effects. The mAb 8E6-mediated complement susceptibility to SZ-Ab61 (ST-229) <span class="html-italic">A. baumannii</span> by guinea pig serum of 100 times dilution; the dose of antibody was 0.5–25 ng. (<b>B</b>) The bactericidal ability of SZ-Ab22 (ST-195) of the complement enhanced by mAb 8E6 (25 ng/well) also appeared in guinea pig serum diluted in 1:100. (<b>C</b>) Both mAbs showed bactericidal ability of the complement and dHL-60 cells against immune strains. Significant differences were detected using the one-way or two-way ANOVA test; *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01; ****, <span class="html-italic">p</span> &lt; 0.0001; ns, not significant versus isotype mAb group. All tests were repeated three times.</p>
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<p>(<b>A</b>) The mAb 1B5- and mAb 8E6-mediated killing heterogenous ST-208 and ST-195 <span class="html-italic">A. baumannii</span> with specificity when combined with guinea pig complement and dHL-60 cells. Each well was consistently replicated in three wells within each experimental cohort; *, <span class="html-italic">p</span> &lt; 0.05 versus isotype mAb group by Tukey’s post hoc test. The complement was diluted 3000 times to achieve a non-specific killing rate (NSK) &lt; 25% in the PBS control. The NSK was calculated as NSK = [1 − (number of colonies in complement control group/number of colonies in inactivated complement control group)] × 100%. (<b>B</b>) The bactericidal rates of mAb 8E6 and mAb 1B5 against 13 strains of antibiotic-resistance <span class="html-italic">A. baumannii</span> were combined, and the median values were marked with “+”, no statistically significant differences (<span class="html-italic">p</span> &gt; 0.05) were observed in the rates of bacterial killing among mAb 8E6 and mAb 1B5.</p>
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<p>The mAbs enhancing the release of immune substances. (<b>A</b>) Mice were infected with ST-208 and ST-229 <span class="html-italic">A. baumannii</span> via nasal intubation drip, and then immediately injected into the mice (n = 10). All mice survived the study period. Significant differences were detected using the Tukey’s Honestly Significant Difference test; *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01; ***, <span class="html-italic">p</span> &lt; 0.001 versus isotype mAb group. All tests were repeated three times. (<b>B</b>) The microscopic architecture of the lungs in mice inoculated with PDR <span class="html-italic">A. baumannii</span> was scrutinized following mAbs protection. The lung tissue was separated at a specified time point and fixed with 4% paraformaldehyde. The rest of the lung tissue homogenates were 10-fold diluted and counted. The sections were stained with hematoxylin and eosin stain and observed under 100× and 400× magnification. More inflammatory cells were observed in the immunized mice during the early stages of the disease. The normal tissue structure recovered more quickly in the immunized mice than in the control group. Inflammatory cells are indicated by yellow pointers, while small focal infiltrations of lymphocytes are represented by blue pointers. Dot infiltrations of lymphocytes and granulocytes are denoted by red pointers, and compensatory increases in the surrounding alveoli are illustrated with gray pointers.</p>
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<p>Mice were inoculated with <span class="html-italic">A. baumannii</span> and subsequently treated with either an antibody or a control treatment (n = 5). Lung tissues were harvested at 4 and 24 h post-infection to assess bacterial load and cytokine levels. (<b>A</b>) No significant disparity in pulmonary bacterial burden was observed between the groups treated with the two distinct antibodies and the control group. (<b>B</b>) The cytokine levels in the lungs of mice administered mAbs exhibited significant variation when compared to the control group; *, <span class="html-italic">p</span> &lt; 0.05 compared to the isotype mAb or the PBS, as determined by the least significant difference (LSD) test.</p>
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<p>(<b>A</b>) Antibodies mediated cross-recognize heterogenous <span class="html-italic">A. baumannii</span> identification. The mAb 8E6 and mAb 1B5 specifically target <span class="html-italic">A. baumannii</span>, as determined using whole-cell ELISA, which was not observed in the isotype mAb; ****, <span class="html-italic">p</span> &lt; 0.0001, versus isotype mAb group by Dunnett’s <span class="html-italic">t</span>-test. (<b>B</b>) Western blot lanes 1, 3, and 5 represent bacterial lysate protein samples SZ-AB57, SZ-AB22, and SZ-AB61, respectively. Meanwhile, lanes 2, 4, and 6 were the lysates of these three bacteria treated with protease K separately. (<b>C</b>) The gray values obtained from the two methods were aggregated and analyzed using a paired <span class="html-italic">t</span>-test; *, <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>LC-MS/MS protein spectrometry results. X-axis represents <span class="html-italic">m</span>/<span class="html-italic">z</span> (mass-to-charge ratio); Y-axis represents relative abundance. The results of co-immunoprecipitation of mAb 1B5 with strain ZH-AB01 are shown in (<b>A</b>), the mAb 1B5 with strain SZ-AB57 are shown in (<b>B</b>); the co-immunoprecipitation of mAb 8E6 with strain ZH-AB01 are shown in (<b>C</b>), the mAb 8E6 with strain SZ-AB57 are shown in (<b>D</b>).</p>
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<p>3D structure of antibody–antigen combination forecast. The light purple color represents the position of the antigen subunit in the macromolecular ATP synthase. Green region is the antibody variable region structure simulated by the LYRA database, and the yellow region is the complementarity determining region identification combined with antigen epitope prediction. (<b>A</b>) The mAb 1B5 adheres to the subunit a. (<b>B</b>) mAb 8E6 adheres to the c ring.</p>
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22 pages, 919 KiB  
Review
Signaling Paradigms of H2S-Induced Vasodilation: A Comprehensive Review
by Constantin Munteanu, Cristina Popescu, Andreea-Iulia Vlădulescu-Trandafir and Gelu Onose
Antioxidants 2024, 13(10), 1158; https://doi.org/10.3390/antiox13101158 - 25 Sep 2024
Viewed by 760
Abstract
Hydrogen sulfide (H2S), a gas traditionally considered toxic, is now recognized as a vital endogenous signaling molecule with a complex physiology. This comprehensive study encompasses a systematic literature review that explores the intricate mechanisms underlying H2S-induced vasodilation. The vasodilatory [...] Read more.
Hydrogen sulfide (H2S), a gas traditionally considered toxic, is now recognized as a vital endogenous signaling molecule with a complex physiology. This comprehensive study encompasses a systematic literature review that explores the intricate mechanisms underlying H2S-induced vasodilation. The vasodilatory effects of H2S are primarily mediated by activating ATP-sensitive potassium (K_ATP) channels, leading to membrane hyperpolarization and subsequent relaxation of vascular smooth muscle cells (VSMCs). Additionally, H2S inhibits L-type calcium channels, reducing calcium influx and diminishing VSMC contraction. Beyond ion channel modulation, H2S profoundly impacts cyclic nucleotide signaling pathways. It stimulates soluble guanylyl cyclase (sGC), increasing the production of cyclic guanosine monophosphate (cGMP). Elevated cGMP levels activate protein kinase G (PKG), which phosphorylates downstream targets like vasodilator-stimulated phosphoprotein (VASP) and promotes smooth muscle relaxation. The synergy between H2S and nitric oxide (NO) signaling further amplifies vasodilation. H2S enhances NO bioavailability by inhibiting its degradation and stimulating endothelial nitric oxide synthase (eNOS) activity, increasing cGMP levels and potent vasodilatory responses. Protein sulfhydration, a post-translational modification, plays a crucial role in cell signaling. H2S S-sulfurates oxidized cysteine residues, while polysulfides (H2Sn) are responsible for S-sulfurating reduced cysteine residues. Sulfhydration of key proteins like K_ATP channels and sGC enhances their activity, contributing to the overall vasodilatory effect. Furthermore, H2S interaction with endothelium-derived hyperpolarizing factor (EDHF) pathways adds another layer to its vasodilatory mechanism. By enhancing EDHF activity, H2S facilitates the hyperpolarization and relaxation of VSMCs through gap junctions between endothelial cells and VSMCs. Recent findings suggest that H2S can also modulate transient receptor potential (TRP) channels, particularly TRPV4 channels, in endothelial cells. Activating these channels by H2S promotes calcium entry, stimulating the production of vasodilatory agents like NO and prostacyclin, thereby regulating vascular tone. The comprehensive understanding of H2S-induced vasodilation mechanisms highlights its therapeutic potential. The multifaceted approach of H2S in modulating vascular tone presents a promising strategy for developing novel treatments for hypertension, ischemic conditions, and other vascular disorders. The interaction of H2S with ion channels, cyclic nucleotide signaling, NO pathways, ROS (Reactive Oxygen Species) scavenging, protein sulfhydration, and EDHF underscores its complexity and therapeutic relevance. In conclusion, the intricate signaling paradigms of H2S-induced vasodilation offer valuable insights into its physiological role and therapeutic potential, promising innovative approaches for managing various vascular diseases through the modulation of vascular tone. Full article
(This article belongs to the Special Issue Hydrogen Sulfide Signaling in Biological Systems)
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<p>This figure illustrates the various molecular and signaling paradigms through which H<sub>2</sub>S contributes to vasodilation: 1. Persulfidation Signaling—a post-translational modification where a sulfur atom is added to the thiol (-SH) group of cysteine residues, forming persulfide (-SSH) groups; 2. K_ATP Channel Activation Paradigm—reduces calcium influx, resulting in smooth muscle relaxation and vasodilation; 3. cGMP Pathway Activation Paradigm—elevated cGMP activates protein kinase G (PKG), which decreases intracellular calcium levels, causing smooth muscle relaxation and vasodilation; 4. Endothelial Nitric Oxide Synthase (eNOS) activation Paradigm; 5. Calcium Signaling Modulation Paradigm; 6. Redox Signaling and Antioxidant Effects Paradigm; 7. Interaction with Other Gasotransmitters Paradigm; 8. Endothelial-Dependent Hyperpolarization (EDH) Paradigm; 9. Interaction with Prostacyclin Signaling Paradigm; 10. Hypoxia Response Paradigm; 11. H<sub>2</sub>S interaction with various transcription factors.</p>
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14 pages, 1619 KiB  
Article
Capsaicin Improves Systemic Inflammation, Atherosclerosis, and Macrophage-Derived Foam Cells by Stimulating PPAR Gamma and TRPV1 Receptors
by Danielle Lima Ávila, Weslley Fernandes-Braga, Janayne Luihan Silva, Elandia Aparecida Santos, Gianne Campos, Paola Caroline Lacerda Leocádio, Luciano Santos Aggum Capettini, Edenil Costa Aguilar and Jacqueline Isaura Alvarez-Leite
Nutrients 2024, 16(18), 3167; https://doi.org/10.3390/nu16183167 - 19 Sep 2024
Viewed by 668
Abstract
Background: Capsaicin, a bioactive compound found in peppers, is recognized for its anti-inflammatory, antioxidant, and anti-lipidemic properties. This study aimed to evaluate the effects of capsaicin on atherosclerosis progression. Methods: Apolipoprotein E knockout mice and their C57BL/6 controls were utilized to assess blood [...] Read more.
Background: Capsaicin, a bioactive compound found in peppers, is recognized for its anti-inflammatory, antioxidant, and anti-lipidemic properties. This study aimed to evaluate the effects of capsaicin on atherosclerosis progression. Methods: Apolipoprotein E knockout mice and their C57BL/6 controls were utilized to assess blood lipid profile, inflammatory status, and atherosclerotic lesions. We also examined the influence of capsaicin on cholesterol influx and efflux, and the role of TRPV1 and PPARγ signaling pathways in bone marrow-derived macrophages. Results: Capsaicin treatment reduced weight gain, visceral adiposity, blood triglycerides, and total and non-HDL cholesterol. These improvements were associated with a reduction in atherosclerotic lesions in the aorta and carotid. Capsaicin also improved hepatic oxidative and inflammatory status. Systemic inflammation was also reduced, as indicated by reduced leukocyte rolling and adhesion on the mesenteric plexus. Capsaicin decreased foam cell formation by reducing cholesterol influx through scavenger receptor A and increasing cholesterol efflux via ATP-binding cassette transporter A1, an effect primarily linked to TRPV1 activation. Conclusions: These findings underscore the potential of capsaicin as a promising agent for atherosclerosis prevention, highlighting its comprehensive role in modulating lipid metabolism, foam cell formation, and inflammatory responses. Full article
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Graphical abstract
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<p>Effects of capsaicin on caloric intake, body composition, glycemia, and lipid profile in WT and ApoE-Deficient mice. Calorie intake (<b>A</b>), weight gain (<b>B</b>), fat percentage (<b>C</b>), glycemia (<b>D</b>), and lipid profile (<b>E</b>–<b>H</b>) of C57BL/6 and Apo E KO animals fed cholesterol-rich diet (AIN-93M + 0.75% cholesterol) without or with 0.015% capsaicin. Bars represent the mean, and vertical lines represent the standard error. Groups not sharing the same letter = statistically different. (<span class="html-italic">p</span> &lt; 0.05). * T Student <span class="html-italic">t</span>-test compared with group without capsaicin. <span class="html-italic">N</span> mice = WT = 5, WT + CAP = 6, ApoE − KO = 11, ApoE + Cap = 10.</p>
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<p>Modulation of Inflammatory Markers by Capsaicin in WT and ApoE KO Mice. Quantification of NAG (<b>A</b>), MPO (<b>B</b>), and the cytokines TNF (<b>C</b>), IL-1β (<b>D</b>), IL-6 (<b>E</b>), and IL-10 (<b>F</b>) in the liver tissue of wild-type animals (C57BL/6) and ApoE KO mice receiving control (AIN-93M + 0.075% cholesterol) or capsaicin-containing (AIN-93M + 0.075% cholesterol + 0.015% capsaicin) diets for 5 weeks. Data are presented as mean (bars) and standard error (vertical lines). Groups not sharing the same letter = statistically different (<span class="html-italic">p</span> &lt; 0.05). <span class="html-italic">N</span> mice = WT = 5, WT + CAP = 6, ApoE − KO = 11, ApoE + Cap = 10, except for NAG and MPO <span class="html-italic">n</span> = 5–6 mice/group.</p>
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<p>Effects of Capsaicin on Atherosclerosis Development and Inflammation (<b>A</b>,<b>B</b>) Percentage of the aorta affected by atherosclerotic lesions in ApoE KO mice after five weeks of ingesting a diet rich in cholesterol with or without Cap (0.015%) <span class="html-italic">n</span> = 8–9 mice/group (<b>C</b>,<b>D</b>)—Atherosclerotic lesion area in the obstructed carotid artery <span class="html-italic">n</span> = 7 mice/group. Fluoresce intensity of monocyte/macrophage (<b>E</b>,<b>F</b>) and CD36 (<b>G</b>,<b>H</b>) staining in the carotid artery (<span class="html-italic">n</span> = 7–10 mice/group). Rolling (<b>I</b>,<b>J</b>) and adhered (<b>K</b>,<b>L</b>) cells in the mesenteric plexus blood by intravital microscopy. Data are presented as mean (bars) and standard error (vertical lines). * Statistically different (<span class="html-italic">p</span> &lt; 0.05). Blue arrows show rolling and adhering leukocytes. <span class="html-italic">N</span> mice = 6–8 mice/group.</p>
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<p>Cholesterol influx and efflux pathways in cultures treated without or with TRPV1 antagonist (CZE (<b>A</b>–<b>E</b>)) or PPARγ inhibitor (<b>F</b>–<b>J</b>). (<b>A</b>)—Total% of oxLDL-Dil positive populations treated or not with CZE; (<b>B</b>)—Total% of SRA-positive cells, treated or not with CZE; (<b>C</b>)—Total% of populations positive for CD36, treated or not with CZE; (<b>D</b>)—Total% of ABCA1-positive cells treated or not with CZE (<b>E</b>)—Total% of ABCG1-positive cells treated or not with CZE; (<b>F</b>)—Total% of oxLDL-Dil positive populations treated or not with GW9662; (<b>G</b>)—Total% of CD36-positive populations, treated or not with GW9662; (<b>H</b>)—Total% of SARS-positive cells, treated or not with GW9662; (<b>I</b>)—Total% of ABCA1-positive cells treated or not with GW9662; (<b>J</b>)—Total% of cells positive for ABCG1 treated or not with GW9662; Bars represent mean and vertical lines represent standard error (SE). Difference statistically significant (<span class="html-italic">p</span> &lt; 0.05) is provided as (*) between each group (with or without inhibitors) and the control group (CT) without inhibitors; (—) between Caps groups treated with inhibitors and the CT group treated with inhibitors; (#) between groups treated with the inhibitor and the same group without the inhibitor.</p>
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12 pages, 9997 KiB  
Article
Molecular Characterization of the MoxR AAA+ ATPase of Synechococcus sp. Strain NKBG15041c
by Kota Mano, Kentaro Noi, Kumiko Oe, Takahiro Mochizuki, Ken Morishima, Rintaro Inoue, Masaaki Sugiyama, Keiichi Noguchi, Kyosuke Shinohara, Masafumi Yohda and Akiyo Yamada
Int. J. Mol. Sci. 2024, 25(18), 9955; https://doi.org/10.3390/ijms25189955 - 15 Sep 2024
Viewed by 638
Abstract
We isolated a stress-tolerance-related gene from a genome library of Synechococcus sp. NKBG15041c. The expression of the gene in E. coli confers resistance against various stresses. The gene encodes a MoxR AAA+ ATPase, which was designated SyMRP since it belongs to the MRP [...] Read more.
We isolated a stress-tolerance-related gene from a genome library of Synechococcus sp. NKBG15041c. The expression of the gene in E. coli confers resistance against various stresses. The gene encodes a MoxR AAA+ ATPase, which was designated SyMRP since it belongs to the MRP subfamily. The recombinant SyMRP showed weak ATPase activity and protected citrate synthase from thermal aggregation. Interestingly, the chaperone activity of SyMRP is ATP-dependent. SyMRP exists as a stable hexamer, and ATP-dependent conformation changes were not detected via analytical ultracentrifugation (AUC) or small-angle X-ray scattering (SAXS). Although the hexameric structure predicted by AlphaFold 3 was the canonical flat-ring structure, the structures observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM) were not the canonical ring structure. In addition, the experimental SAXS profiles did not show a peak that should exist in the symmetric-ring structure. Therefore, SyMRP seems to form a hexameric structure different from the canonical hexameric structure of AAA+ ATPase. Full article
(This article belongs to the Section Molecular Microbiology)
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<p>Amino acid sequence alignment (<b>a</b>) and phylogenetic tree of SyMRP (<b>b</b>). Amino acid sequence alignments were performed via ClustalOmega. A phylogenetic tree was generated by iTOL.</p>
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<p>Effects of SyMRP on the growth of <span class="html-italic">E. coli</span> under various stress conditions. (<b>a</b>–<b>d</b>) Growth curves under various stress conditions. (<b>a</b>) Nonstress condition. (<b>b</b>) Salt stress condition (1.0 M NaCl). (<b>c</b>) Acid stress condition (pH 4.0). (<b>d</b>) Thermal stress condition (45 °C). Red circle: pSK-SyMRP; blue circle: pSK-BAA10517; black: vector control. (<b>e</b>–<b>g</b>) Spot tests. 1: pSK-SyMRP, 2: pSK-BAA10517, 3: vector control. (<b>e</b>) Nonstress condition. (<b>f</b>) Salt stress condition (0.8 M NaCl). (<b>g</b>) Thermal stress condition (50 °C, 30 min).</p>
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<p>Effects of SyMRP on the growth of <span class="html-italic">E. coli</span> under various stress conditions. (<b>a</b>–<b>d</b>) Growth curves under various stress conditions. (<b>a</b>) Nonstress condition. (<b>b</b>) Salt stress condition (1.0 M NaCl). (<b>c</b>) Acid stress condition (pH 4.0). (<b>d</b>) Thermal stress condition (45 °C). Red circle: pSK-SyMRP; blue circle: pSK-BAA10517; black: vector control. (<b>e</b>–<b>g</b>) Spot tests. 1: pSK-SyMRP, 2: pSK-BAA10517, 3: vector control. (<b>e</b>) Nonstress condition. (<b>f</b>) Salt stress condition (0.8 M NaCl). (<b>g</b>) Thermal stress condition (50 °C, 30 min).</p>
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<p>Chaperone function of SyMRP. The thermal aggregation of CS from the porcine heart was monitored by measuring light scattering at 500 nm with a spectrofluorometer at 43 °C. CS (0.1 µM, monomer) was incubated in assay buffer with or without SyMRP and BSA at the specified concentration. To examine their effects, 1 mM ATP (<b>a</b>) or ADP (<b>b</b>) was added.</p>
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<p>AUC results of SyMRP solutions at 25 °C. The red and blue lines show the concentration distributions of molecules as a function of the sedimentation coefficient for the solutions without and with ATP, respectively.</p>
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<p>SAXS profiles of SyMRP solutions at 25 °C. The red and blue circles show the SAXS profiles of the solutions without and with ATP, respectively. The solid black line indicates the calculated scattering profile for the hexameric model predicted by AlphaFold 3. The arrow represents the peak for the calculated scattering profile at <span class="html-italic">q</span> ~ 0.2 Å<sup>−1</sup>.</p>
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<p>Hexameric structure of SyMRP predicted by AlphaFold 3. (<b>a</b>) Top view, (<b>b</b>) side view.</p>
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<p>AFM and TM images of SyMRP. (<b>a</b>) Images of small oligomers observed by AFM. (<b>b</b>) Image of probable hexamers observed by TEM (<b>top</b>) and AFM (<b>bottom</b>).</p>
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25 pages, 3059 KiB  
Article
Discovery of Benzopyrone-Based Candidates as Potential Antimicrobial and Photochemotherapeutic Agents through Inhibition of DNA Gyrase Enzyme B: Design, Synthesis, In Vitro and In Silico Evaluation
by Akram Abd El-Haleem, Usama Ammar, Domiziana Masci, Sohair El-Ansary, Doaa Abdel Rahman, Fatma Abou-Elazm and Nehad El-Dydamony
Pharmaceuticals 2024, 17(9), 1197; https://doi.org/10.3390/ph17091197 - 11 Sep 2024
Viewed by 599
Abstract
Bacterial DNA gyrase is considered one of the validated targets for antibacterial drug discovery. Benzopyrones have been reported as promising derivatives that inhibit bacterial DNA gyrase B through competitive binding into the ATP binding site of the B subunit. In this study, we [...] Read more.
Bacterial DNA gyrase is considered one of the validated targets for antibacterial drug discovery. Benzopyrones have been reported as promising derivatives that inhibit bacterial DNA gyrase B through competitive binding into the ATP binding site of the B subunit. In this study, we designed and synthesized twenty-two benzopyrone-based derivatives with different chemical features to assess their antimicrobial and photosensitizing activities. The antimicrobial activity was evaluated against B. subtilis, S. aureus, E. coli, and C. albicans. Compounds 6a and 6b (rigid tetracyclic-based derivatives), 7a-7f (flexible-linker containing benzopyrones), and 8a-8f (rigid tricyclic-based compounds) exhibited promising results against B. subtilis, S. aureus, and E. coli strains. Additionally, these compounds demonstrated photosensitizing activities against the B. subtilis strain. Both in silico molecular docking and in vitro DNA gyrase supercoiling inhibitory assays were performed to study their potential mechanisms of action. Compounds 8a-8f exhibited the most favorable binding interactions, engaging with key regions within the ATP binding site of the DNA gyrase B domain. Moreover, compound 8d displayed the most potent IC50 value (0.76 μM) compared to reference compounds (novobiocin = 0.41 μM and ciprofloxacin = 2.72 μM). These results establish a foundation for structure-based optimization targeting DNA gyrase inhibition with antibacterial activity. Full article
(This article belongs to the Special Issue In Silico and In Vitro Screening of Small Molecule Inhibitors)
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<p>Diagrammatic illustration of the molecular structure of DNA gyrase enzyme and the function of each component. DNA gyrase is a tetrameric enzyme composed of two GyrA and two GyrB subunits (GyrA2GyrB2). Structurally, this complex is organized through three sets of “gates”, whose sequential opening and closing facilitate the direct transfer of DNA segments and the introduction of two negative supercoils. The N-gates are shaped by the ATPase domains of the GyrB subunits. The binding of two ATP molecules triggers dimerization, leading to the closure of these gates. Conversely, hydrolysis causes their opening. The catalytic center responsible for DNA cleavage and reunion is situated in the DNA gates formed by all gyrase subunits. On the other hand, the C-gates are formed by the GyrA subunits [<a href="#B13-pharmaceuticals-17-01197" class="html-bibr">13</a>,<a href="#B14-pharmaceuticals-17-01197" class="html-bibr">14</a>].</p>
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<p>The reported antimicrobial agents (DNA gyrase inhibitors, (<b>A</b>)) and active photochemotherapeutic furobenzopyrones (<b>B</b>).</p>
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<p>The rational design of first-in-class benzopyrone-based candidates as dual antimicrobial and photochemotherapeutic agents. A group of structural modifications and developments has been applied to the newly designed benzopyrone-based derivatives. We used both reported benzopyrone derivatives (novobiocin and clorobiocin) and benzothiazole derivative (<b>II</b>) as lead compounds to build our compounds. The fused bicyclic benzopyrone ring was selected to be the central scaffold in our designed derivatives. The phenyl alkyl amide group of benzothiazole derivative (<b>II</b>) was replaced with rigid benzopyrone isostere to maintain the bicyclic structure and incorporate a HBA group in order to provide H-bond interactions with the key amino acid residues within the ATP active site of DNA gyrase B. The fused five-membered thiazole ring of benzothiazole derivative (<b>II</b>) was replaced with its isostere, furan ring, to mimic the photochemotherapeutic agents. Accordingly, a novel series of rigid-based derivatives were designed with the angular furobenzopyrone scaffold in their core structures. An additional phenyl ring was incorporated in fused form (tetracyclic-based derivatives, <b>6a</b> and <b>6b</b>) and substituted form (tricyclic-based compounds, <b>8a</b>-<b>8f</b>) to evaluate accessibility into the active site of the gyrase B domain. Additional series with flexible linkers between the benzopyrone core scaffold and the distal phenyl ring were designed (<b>7a</b>-<b>7f</b>) to evaluate the effect of HBA groups on the binding affinity into the active site.</p>
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<p>Photosensitizing activity of tested compounds against <span class="html-italic">B. subtilis</span> expressed as minimum inhibitory concentrations (MIC, mg mL<sup>−1</sup>) using xanthotoxin as standard. MIC of tested compounds before and after irradiation with UV lamp (360 nm) for 20 min.</p>
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<p>3D binding interactions of generated conformational clusters of the tested compounds (<b>6b</b>, <b>7a</b>, and <b>8d</b>) and the reference ligand (<b>II</b>) within the ATP binding site of the <span class="html-italic">N</span>-terminal domain of gyrase B subunit (GyrB24kDa, PDB ID: 6YD9). (<b>A</b>), the reference ligand (<b>II</b>), showed a key H-binding interaction with Arg136 (2.1 Å) and hydrophobic interactions with the valine-rich region (Val43, Val71, Val120, and Val167); (<b>B</b>), the rigid tetracyclic benzopyrone derivative (<b>6b</b>), showed H-bond interaction with Gly77 (2.2 Å). The fused phenyl ring could not access the valine-rich hydrophobic pocket (left region) to exhibit key hydrophobic interactions; (<b>C</b>), the flexible-spacer-containing compound (<b>7a</b>), showed a key H-bond interaction with Gly77 (2.0 Å). The rotation of the distal phenyl ring disrupted the coplanarity of the structure, bringing the distal phenyl ring away from the valine-rich hydrophobic pocket; (<b>D</b>), the rigid tricyclic benzopyrone derivative (<b>8e</b>), showed the highest docking score and the greatest number of binding interactions among the tested compounds and the reference ligand (<b>II</b>). It displayed a strong H-bond interaction with Thr165 (2.1 Å). The distal phenyl ring, along with the 4-Me group, was oriented to fully access the valine-rich hydrophobic pocket.</p>
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<p>General molecular interactions between the designed compounds (rigid tricyclic derivatives, <b>8d</b>-<b>8f</b>) and the ATP binding site of the gyrase B subunit. R = H (<b>8d</b>), Me (<b>8e</b>), OMe (<b>8f</b>). The diagram illustrates the key regions within the ATP binding site. The central benzopyrone core scaffold exhibited a number of hydrophobic interactions with the hydrophobic surface (black). The HBA (CO) of benzopyrone showed strong HB interactions with Gly77 or Thr165 (&lt;2.3 Å, purple). The phenyl ring is positioned at the solvent-exposed area, showing a number of hydrophobic interactions with the key amino acid residues at this region (green). The distal phenyl ring displayed different hydrophobic interactions with the valine-rich hydrophobic pocket (red). The rigid angular furan ring forced the distal phenyl ring into the valine-rich hydrophobic pocket, modulating the coplanarity of the designed compounds.</p>
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<p>Reagents and conditions. <b>a</b>, 3-chlorobutan-2-one, acetone, K<sub>2</sub>CO<sub>3</sub>, reflux, 24 h; <b>b</b>, NaOH, iPrOH, reflux, 4 h; <b>c</b>, 2-chlorocyclohexanone, acetone, K<sub>2</sub>CO<sub>3</sub> reflux, 24 h; <b>d</b>, NaOH, iPrOH, reflux, 4 h; <b>e</b>, DDQ, benzene, reflux, 20 h.</p>
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<p>Reagents and conditions. <b>a</b>, appropriate phenacyl bromide, acetone, K<sub>2</sub>CO<sub>3</sub>, reflux, 24 h; <b>b</b>, Na, EtOH, 2 h.</p>
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14 pages, 2352 KiB  
Article
Bisphenol S Promotes the Transfer of Antibiotic Resistance Genes via Transformation
by Jiayi Zhang, Shuyao Zhu, Jingyi Sun and Yuan Liu
Int. J. Mol. Sci. 2024, 25(18), 9819; https://doi.org/10.3390/ijms25189819 - 11 Sep 2024
Viewed by 627
Abstract
The antibiotic resistance crisis has seriously jeopardized public health and human safety. As one of the ways of horizontal transfer, transformation enables bacteria to acquire exogenous genes naturally. Bisphenol compounds are now widely used in plastics, food, and beverage packaging, and have become [...] Read more.
The antibiotic resistance crisis has seriously jeopardized public health and human safety. As one of the ways of horizontal transfer, transformation enables bacteria to acquire exogenous genes naturally. Bisphenol compounds are now widely used in plastics, food, and beverage packaging, and have become a new environmental pollutant. However, their potential relationship with the spread of antibiotic resistance genes (ARGs) in the environment remains largely unexplored. In this study, we aimed to assess whether the ubiquitous bisphenol S (BPS) could promote the transformation of plasmid-borne ARGs. Using plasmid pUC19 carrying the ampicillin resistance gene as an extracellular ARG and model microorganism E. coli DH5α as the recipient, we established a transformation system. Transformation assays revealed that environmentally relevant concentrations of BPS (0.1–10 μg/mL) markedly enhanced the transformation frequency of plasmid-borne ARGs into E. coli DH5α up to 2.02-fold. Fluorescent probes and transcript-level analyses suggest that BPS stimulated increased reactive oxygen species (ROS) production, activated the SOS response, induced membrane damage, and increased membrane fluidity, which weakened the barrier for plasmid transfer, allowing foreign DNA to be more easily absorbed. Moreover, BPS stimulates ATP supply by activating the tricarboxylic acid (TCA) cycle, which promotes flagellar motility and expands the search for foreign DNA. Overall, these findings provide important insight into the role of bisphenol compounds in facilitating the horizontal spread of ARGs and emphasize the need to monitor the residues of these environmental contaminants. Full article
(This article belongs to the Section Molecular Microbiology)
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<p>BPS promotes the transformation of ARGs into <span class="html-italic">E. coli</span> DH5α. (<b>A</b>) Growth curves of the recipient bacterium (<span class="html-italic">E. coli</span> DH5α) in the presence of different concentrations of the BPS (0.1–10 μg/mL). (<b>B</b>) Effects of different concentrations of the BPS on the frequency of transformation of pUC19 plasmid into <span class="html-italic">E. coli</span> DH5α. Statistically significant differences were determined using one-way ANOVA at * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001, respectively. NS, not significant. (<b>C</b>) Gel electropherograms of pUC19 plasmid, recipient bacteria, and transformants at different concentrations of BPS. (<b>D</b>) MIC values of recipient bacteria and transformants.</p>
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<p>BPS stimulates the production of ROS and enhances membrane permeability in the recipient bacteria. (<b>A</b>) Effects of different concentrations of BPS on ROS production by recipient bacteria. (<b>B</b>) Heat map of increased expression levels of genes related to the oxidative stress system and SOS response system of bacteria after BPS treatment. (<b>C</b>) Changes in outer membrane permeability of recipient bacteria following BPS pressure. (<b>D</b>) Changes in inner membrane permeability in response to BPS treatment. (<b>E</b>) Effect of BPS on membrane fluidity. Statistically significant differences were determined using one-way ANOVA at * <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 and **** <span class="html-italic">p</span> &lt; 0.0001, respectively. NS, not significant. (<b>F</b>) Heatmap of the increased expression levels of genes related to bacterial membrane permeability after BPS treatment. (<b>G</b>) SEM images of <span class="html-italic">E. coli</span> DH5α bacterial cells exposed to 0.5 μg/mL BPS for 4 h. Cell membrane damage is indicated by red arrows.</p>
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<p>BPS enhances bacterial metabolism by accelerating the TCA cycle. (<b>A</b>) Bacterial respiration levels of <span class="html-italic">E. coli</span> DH5α were unchanged or even decreased under the pressure of BPS. (<b>B</b>) Heatmap of the expression levels of genes related to bacterial electron transport chain in response to BPS treatment. (<b>C</b>) Heatmap of TCA cycle-related gene expression levels in response to BPS. Bacterial (<b>D</b>) NAD<sup>+</sup>/NADH ratio, (<b>E</b>) NAD<sup>+</sup> content, and (<b>F</b>) NADH content under BPS treatment. Statistically significant differences were determined using one-way ANOVA at ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 and **** <span class="html-italic">p</span> &lt; 0.0001, respectively. NS, not significant.</p>
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<p>BPS stimulates ATP synthesis and flagellar motility. (<b>A</b>) ΔpH changes of recipient bacteria in response to BPS treatment, measured using BCECF. (<b>B</b>) Membrane potential of recipient bacteria in response to BPS stress, monitored using DiSC<sub>3</sub>(5). (<b>C</b>) Bacterial ATP synthesis after exposure to BPS. (<b>D</b>) Heat map of the expression level of bacterial ATP synthase-related genes under BPS stress. (<b>E</b>) Heatmap of the expression level of bacterial flagellum-related genes after BPS treatment. (<b>F</b>) Swimming motility test of <span class="html-italic">E. coli</span> DH5α under BPS stress, scale bar, 0.5 cm. Statistically significant differences were determined using one-way ANOVA at **** <span class="html-italic">p</span> &lt; 0.0001. NS, not significant.</p>
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<p>Schematic diagram of the mechanism of increased transformation by BPS treatment. The frequency of transformation of antibiotic-resistant plasmids was significantly increased under the stress of low concentrations of BPS. Potential mechanisms include a dramatic increase in ROS production and activation of the SOS response, which increases membrane permeability and fluidity. In addition, the accelerated TCA cycle generates a large amount of ATP, and flagellar motility was also enhanced. These actions are favorable for plasmid uptake, facilitation, and integration.</p>
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18 pages, 2318 KiB  
Article
The Archetypal Gamma-Core Motif of Antimicrobial Cys-Rich Peptides Inhibits H+-ATPases in Target Pathogens
by María T. Andrés, Nannette Y. Yount, Maikel Acosta-Zaldívar, Michael R. Yeaman and José F. Fierro
Int. J. Mol. Sci. 2024, 25(17), 9672; https://doi.org/10.3390/ijms25179672 - 6 Sep 2024
Viewed by 537
Abstract
Human lactoferrin (hLf) is an innate host defense protein that inhibits microbial H+-ATPases. This protein includes an ancestral structural motif (i.e., γ-core motif) intimately associated with the antimicrobial activity of many natural Cys-rich peptides. Peptides containing a complete γ-core motif from [...] Read more.
Human lactoferrin (hLf) is an innate host defense protein that inhibits microbial H+-ATPases. This protein includes an ancestral structural motif (i.e., γ-core motif) intimately associated with the antimicrobial activity of many natural Cys-rich peptides. Peptides containing a complete γ-core motif from hLf or other phylogenetically diverse antimicrobial peptides (i.e., afnA, SolyC, PA1b, PvD1, thanatin) showed microbicidal activity with similar features to those previously reported for hLf and defensins. Common mechanistic characteristics included (1) cell death independent of plasma membrane (PM) lysis, (2) loss of intracellular K+ (mediated by Tok1p K+ channels in yeast), (3) inhibition of microbicidal activity by high extracellular K+, (4) influence of cellular respiration on microbicidal activity, (5) involvement of mitochondrial ATP synthase in yeast cell death processes, and (6) increment of intracellular ATP. Similar features were also observed with the BM2 peptide, a fungal PM H+-ATPase inhibitor. Collectively, these findings suggest host defense peptides containing a homologous γ-core motif inhibit PM H+-ATPases. Based on this discovery, we propose that the γ-core motif is an archetypal effector involved in the inhibition of PM H+-ATPases across kingdoms of life and contributes to the in vitro microbicidal activity of Cys-rich antimicrobial peptides. Full article
(This article belongs to the Collection Feature Papers in Molecular Immunology)
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<p>Cell viability and membrane permeabilization assays. <span class="html-italic">C. albicans</span> or <span class="html-italic">P. aeruginosa</span> (10<sup>6</sup> cells/mL) were incubated (90 min, 37 °C) with each peptide/protein at the indicated concentrations. Aliquots were plated on SDA or TSB, and colonies were counted after 24–48 h (<span class="html-italic">C. albicans</span>) or 24 h (<span class="html-italic">P. aeruginosa</span>). At each point, samples were stained with propidium iodide (PI) and intracellular PI incorporation, indicative of membrane permeabilization, was quantified by flow cytometry. The relative cell viability (blue lines) and cellular permeabilization (red lines) were calculated as the percentage of untreated cells. Human lactoferrin (hLf) and the peptide Lfpep were used as negative and positive controls of cellular permeabilization, respectively. The results are the means of at least three independent experiments, and error bars represent standard deviations (±SD).</p>
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<p>Potassium efflux induced by hLf γ-core-containing peptides. (<b>A</b>,<b>D</b>) Kinetics of K<sup>+</sup> efflux. <span class="html-italic">C. albicans</span> or <span class="html-italic">P. aeruginosa</span> (10<sup>7</sup> cells/mL) were incubated with kaliocin-1, Kdp15, human lactoferrin (hLf), and Lfpep. hLf and Lfpep were used as negative and positive controls of the membrane permeabilization, respectively. The effect of antifungal BM2 peptide was evaluated. The extracellular K<sup>+</sup> concentration was quantified at different times. (<b>B</b>,<b>C</b>,<b>E</b>) Effect of extracellular K<sup>+</sup> and TEA<sup>+</sup> on microbicidal activity. (<b>B</b>,<b>E</b>) microbicidal activity was assessed in Tris buffer in the absence or in the presence of 50 mM KCl or (<b>C</b>) 10 mM TEA<sup>+</sup>. Cell viability was determined by plating aliquots of the cell suspensions. The percentage of viable cells was determined relative to that for cells incubated without peptides (control). Results are the mean ± SD from duplicates of at least three independent determinations. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 were used.</p>
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<p>Effect of cellular respiration on the microbicidal activity of hLf γ-core-containing peptides. (<b>A</b>,<b>C</b>) Oxygen consumption was measured in <span class="html-italic">C. albicans</span> and <span class="html-italic">P. aeruginosa</span> cellular suspensions in the absence (control) or the presence of kaliocin-1 (Kal-1), Kdp15, or human lactoferrin (hLf), and piericidin A (positive control). (<b>B</b>,<b>D</b>) Effect of cellular respiration on the microbicidal activity of hLf γ-core-containing peptides. Cells (10<sup>6</sup> cells/mL) were pre-incubated (15 min, 37 °C) with 32 μM piericidin A and incubated for 90 min at 37 °C with the peptides hLf or BM2. The cell viability was determined by means of the plate counting method. The results are the mean ± SD from duplicates of at least three independent experiments. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 were used.</p>
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<p>Effect of inhibition of mitochondrial ATP synthase on the candidacidal activity of hLf γ-core-containing peptides. Candidacidal effect of IC<sub>50</sub> of kaliocin-1 (Kal-1) and Kdp15 and 0.25 μM BM2 on cells (10<sup>6</sup> cells/mL) pre-incubated without or with 16 μg/mL oligomycin A. Cell viability was determined by plating aliquots of the cell suspensions and the percentage of viable cells was determined relative to that for cells incubated without peptides. Human lactoferrin (hLf) and Lfpep were used as positive and negative controls, respectively. Data are the mean ± SD from at least three experiments. ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 were used.</p>
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<p>Effect of hLf γ-core-containing peptides on PM H<sup>+</sup>-ATPases. (<b>A</b>) Effect of hLf γ-core-containing peptides on glucose-dependent external acidification. <span class="html-italic">C. albicans</span> cells (10<sup>7</sup> cells/mL) in 50 mM KCl were preincubated with the peptides (2 × IC<sub>50</sub>) for 20 min. Glucose (final concentration of 2.5 mM) was then added to induce H<sup>+</sup> efflux via Pma1p H<sup>+</sup>-ATPase, as indicated by the subsequent external acidification. For clarity, only one set of three independent experiments and values measured every 5 min are represented. (<b>B</b>) Effect of hLf γ-core-containing peptides on intracellular ATP (iATP) levels. Measurement of iATP of <span class="html-italic">C. albicans</span> cells (<b>B</b>) cells in the presence of kaliocin-1 (Kal-1) or Kdp15. The PM H<sup>+</sup>-ATPases inhibitors, human lactoferrin (hLf), and the peptide BM2 were used as positive controls. The total cellular iATP concentrations were determined after 90 min of treatment. The mean data ± SD are indicated as the percent change compared to that for the untreated control sample.</p>
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<p>Effect of different inhibitors on microbicidal activity of phylogenetically diverse γ-core-containing peptides. (<b>A</b>,<b>D</b>) Microbicidal effect of different γ-core-containing peptides. Cell suspensions (10<sup>6</sup> cells/mL) in Tris buffer were incubated for 90 min at 37 °C with the peptides and cell viability was determined by a plating counting method. (<b>B</b>,<b>E</b>) Effect of different inhibitors on microbicidal activity. (<b>C</b>) Effect of different γ-core-containing peptides on glucose-dependent external acidification by <span class="html-italic">C. albicans</span>. For clarity, only one set of three independent experiments and values measured every 5 min are represented. (<b>F</b>) Effects of different γ-core-containing peptides on membrane integrity. Bars represent the percentage of propidium iodide (PI)-positive cells incubated with the peptides. The results are the mean ± SD from duplicates of at least three independent experiments.</p>
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32 pages, 1923 KiB  
Review
The Spectrum of Disease-Associated Alleles in Countries with a Predominantly Slavic Population
by Grigoriy A. Yanus, Evgeny N. Suspitsin and Evgeny N. Imyanitov
Int. J. Mol. Sci. 2024, 25(17), 9335; https://doi.org/10.3390/ijms25179335 - 28 Aug 2024
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Abstract
There are more than 260 million people of Slavic descent worldwide, who reside mainly in Eastern Europe but also represent a noticeable share of the population in the USA and Canada. Slavic populations, particularly Eastern Slavs and some Western Slavs, demonstrate a surprisingly [...] Read more.
There are more than 260 million people of Slavic descent worldwide, who reside mainly in Eastern Europe but also represent a noticeable share of the population in the USA and Canada. Slavic populations, particularly Eastern Slavs and some Western Slavs, demonstrate a surprisingly high degree of genetic homogeneity, and, consequently, remarkable contribution of recurrent alleles associated with hereditary diseases. Along with pan-European pathogenic variants with clearly elevated occurrence in Slavic people (e.g., ATP7B c.3207C>A and PAH c.1222C>T), there are at least 52 pan-Slavic germ-line mutations (e.g., NBN c.657_661del and BRCA1 c.5266dupC) as well as several disease-predisposing alleles characteristic of the particular Slavic communities (e.g., Polish SDHD c.33C>A and Russian ARSB c.1562G>A variants). From a clinical standpoint, Slavs have some features of a huge founder population, thus providing a unique opportunity for efficient genetic studies. Full article
(This article belongs to the Section Molecular Genetics and Genomics)
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Figure 1

Figure 1
<p>Historical dispersal of Slavic people. Approximate time of migration (years CE) is given in brackets.</p>
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<p>Minor allele frequency (MAF) of the <span class="html-italic">NBN</span> c.657del5 allele in Slavic and some non-Slavic countries (created with <a href="http://paintmaps.com" target="_blank">paintmaps.com</a>, accessed on date 27 May 2024).</p>
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