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Stresses, Volume 4, Issue 3 (September 2024) – 11 articles

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23 pages, 6157 KiB  
Article
Stomatal and Non-Stomatal Leaf Responses during Two Sequential Water Stress Cycles in Young Coffea canephora Plants
by Danilo F. Baroni, Guilherme A. R. de Souza, Wallace de P. Bernado, Anne R. Santos, Larissa C. de S. Barcellos, Letícia F. T. Barcelos, Laísa Z. Correia, Claudio M. de Almeida, Abraão C. Verdin Filho, Weverton P. Rodrigues, José C. Ramalho, Miroslava Rakočević and Eliemar Campostrini
Stresses 2024, 4(3), 575-597; https://doi.org/10.3390/stresses4030037 - 9 Sep 2024
Viewed by 440
Abstract
Understanding the dynamics of physiological changes involved in the acclimation responses of plants after their exposure to repeated cycles of water stress is crucial to selecting resilient genotypes for regions with recurrent drought episodes. Under such background, we tried to respond to questions [...] Read more.
Understanding the dynamics of physiological changes involved in the acclimation responses of plants after their exposure to repeated cycles of water stress is crucial to selecting resilient genotypes for regions with recurrent drought episodes. Under such background, we tried to respond to questions as: (1) Are there differences in the stomatal-related and non-stomatal responses during water stress cycles in different clones of Coffea canephora Pierre ex A. Froehner? (2) Do these C. canephora clones show a different response in each of the two sequential water stress events? (3) Is one previous drought stress event sufficient to induce a kind of “memory” in C. canephora? Seven-month-old plants of two clones (’3V’ and ‘A1’, previously characterized as deeper and lesser deep root growth, respectively) were maintained well-watered (WW) or fully withholding the irrigation, inducing soil water stress (WS) until the soil matric water potential (Ψmsoil) reached ≅ −0.5 MPa (−500 kPa) at a soil depth of 500 mm. Two sequential drought events (drought-1 and drought-2) attained this Ψmsoil after 19 days and were followed by soil rewatering until a complete recovery of leaf net CO2 assimilation rate (Anet) during the recovery-1 and recovery-2 events. The leaf gas exchange, chlorophyll a fluorescence, and leaf reflectance parameters were measured in six-day frequency, while the leaf anatomy was examined only at the end of the second drought cycle. In both drought events, the WS plants showed reduction in stomatal conductance and leaf transpiration. The reduction in internal CO2 diffusion was observed in the second drought cycle, expressed by increased thickness of spongy parenchyma in both clones. Those stomatal and anatomical traits impacted decreasing the Anet in both drought events. The ‘3V’ was less influenced by water stress than the ‘A1’ genotype in Anet, effective quantum yield in PSII photochemistry, photochemical quenching, linear electron transport rate, and photochemical reflectance index during the drought-1, but during the drought-2 event such an advantage disappeared. Such physiological genotype differences were supported by the medium xylem vessel area diminished only in ‘3V’ under WS. In both drought cycles, the recovery of all observed stomatal and non-stomatal responses was usually complete after 12 days of rewatering. The absence of photochemical impacts, namely in the maximum quantum yield of primary photochemical reactions, photosynthetic performance index, and density of reaction centers capable of QA reduction during the drought-2 event, might result from an acclimation response of the clones to WS. In the second drought cycle, the plants showed some improved responses to stress, suggesting “memory” effects as drought acclimation at a recurrent drought. Full article
(This article belongs to the Topic Plant Responses to Environmental Stress)
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Graphical abstract

Graphical abstract
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<p>Soil matric water potential (Ψ<sub>msoil</sub>) at 100 mm cm and 500 mm from the soil surface in the pots of the <span class="html-italic">C. canephora</span> var. Robusta genotypes of (<b>A</b>) ‘3V’ and (<b>B</b>) ‘A1’ under well-watered (WW) and water stressed (WS) conditions. The water restriction was imposed during the drought-1 and drought-2 events, after which the soil was rewatered (and recovery-1 and recovery-2 events).</p>
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<p>Leaf gas exchanges of two genotypes (Gen) of <span class="html-italic">C. canephora</span> var. Robusta (‘3V’ and ‘A1’) grown under two water availability conditions [Wat, well-watered (WW) and water stress (WS)], over 12 time-points of six-day intervals (Day) during drought-1 and drought-2 and respective recovery events: (<b>A</b>) net CO<sub>2</sub> assimilation rate (<span class="html-italic">A</span><sub>net</sub>), (<b>B</b>) stomatal conductance to water (<span class="html-italic">g</span><sub>s</sub>), (<b>C</b>) transpiration rate (<span class="html-italic">E</span>), and (<b>D</b>) leaf-to-air vapor pressure deficit (VPD<sub>leaf-air</sub>). Inside the figures, the different lowercase letters indicate the significant difference among the time-points for each water regime (blue for WW and olive green for WS); different uppercase letters indicate the comparison between water availabilities for each time-point of observation (blue for WW and olive green for WS); and different superscript black ■ signs indicate that ‘3V’ was statistically superior to ‘A1’ at that time-point. Mean ± SE and ANOVA <span class="html-italic">p</span>-values (n = 7) for effects of three factors (water availability, genotype, and day of observation) and their interactions are shown. The significant <span class="html-italic">p</span>-values were marked in bold in the upper part of each graph.</p>
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<p>Instantaneous water-use efficiency (WUE, <span class="html-italic">A</span><sub>net</sub>/<span class="html-italic">E</span>) of two genotypes (Gen) of <span class="html-italic">C. canephora</span> var. Robusta (‘3V’ and ‘A1’) grown under two water availability conditions [Wat, well-watered (WW) and water stress (WS)], over 12 time-points of six-day intervals (Day) during drought-1 and drought-2 and respective recovery events. Inside the figure, different lowercase letters indicate the significant difference among the day-time points for each water regime (blue for WW and olive green for WS); different uppercase letters indicate the comparison between water availabilities for each time-point of observation (blue for WW and olive green for WS). Mean ± SE and ANOVA <span class="html-italic">p</span>-values (n = 7) for effects of three factors (water availability, genotype, and day of observation) and their interactions are shown. The significant <span class="html-italic">p</span>-values were marked in bold in the upper part of each graph.</p>
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<p>Variation of OJIP indexes of two genotypes (Gen) of <span class="html-italic">C. canephora</span> var. Robusta (‘3V’ and ‘A1’) grown under two water availability conditions [Wat, well-watered (WW) and water stress (WS)] over 12 time-points of six-day intervals (Day) during drought-1 and drought-2 and respective recovery events: (<b>A</b>) maximum quantum yield of primary photochemical reactions (ΦP<sub>0</sub>), (<b>B</b>) probability of electron transfer from Q<sub>A</sub>-to-electron transport chain beyond Q<sub>A</sub> (ΨE<sub>0</sub>), (<b>C</b>) photosynthetic performance index (PI<sub>ABS</sub>), and (<b>D</b>) density of reaction centers capable of Q<sub>A</sub> reduction (RC/CS<sub>0</sub>). Inside the figures, the different lowercase letters indicate the significant difference among the time-points for each water regime (blue for WW and olive green for WS); different uppercase letters indicate the comparison between water availabilities for each day of observation (blue for WW and olive green for WS); superscript black ■ signs indicate that ‘3V’ was statistically superior to ‘A1’, while superscript black ● signs indicate that ‘A1’ clone was statistically superior to ‘3V’ clone at that time-point. Mean ± SE and ANOVA <span class="html-italic">p</span>-values (n = 7) for effects of three factors (water availability, genotype, and day of observation) and their interactions are shown. The significant <span class="html-italic">p</span>-values were marked in bold.</p>
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<p>Variation of modulated chlorophyll <span class="html-italic">a</span> fluorescence indexes of two genotypes (Gen) of <span class="html-italic">C. canephora</span> var. Robusta (‘3V’ and ‘A1’) grown under two water availability conditions [Wat, well-watered (WW) and water stress (WS)] over 12 time-points of six-day intervals (Day) during drought-1 and drought-2 and respective recovery events: (<b>A</b>) effective quantum yield in PSII photochemistry (Φ<sub>PSII</sub>), (<b>B</b>) photochemical quenching (qP), (<b>C</b>) non-photochemical quenching (NPQ), and (<b>D</b>) linear electron transport rate (ETR). Inside the figures, the different lowercase letters indicate the significant difference among the time-points for each water regime (blue for WW and olive green for WS); different uppercase letters indicate the comparison between water availabilities for each day of observation (blue for WW and olive green for WS); different superscript black ■ signs indicate that ‘3V’ was statistically superior to ‘A1’, while superscript black ● signs indicate that ‘A1’ clone was statistically superior to ‘3V’ clone at that time-point. Mean ± SE and ANOVA <span class="html-italic">P</span>-values (n = 7) for effects of three factors (water availability, genotype, and day of observation) and their interactions are shown. The significant <span class="html-italic">P</span>-values were marked in bold.</p>
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<p>Variation of spectral reflectance indices of leaf adaxial surface of two genotypes (Gen) of C. canephora var. Robusta (‘3V’ and ‘A1’) grown under two water availability conditions [Wat, well-watered (WW) and water stress (WS)] over 12 time-points of six-day intervals (Day) during drought-1 and drought-2 and respective recovery events: (<b>A</b>) green chlorophyll index (GCI), (<b>B</b>) carotenoid reflectance index (CRI), (<b>C</b>) photochemical reflectance index (PRI), and (<b>D</b>) structure intensive reflectance index (SIPI). Inside the figures, the different lowercase letters indicate the significant difference among the time-points for each water regime (blue for WW and olive green for WS); different uppercase letters indicate the comparison between water availabilities for each day of observation (blue for WW and olive green for WS); different superscript black ■ signs indicate that ‘3V’ was statistically superior to ‘A1’, while superscript black ● signs indicate that ‘A1’ clone was statistically superior to ‘3V’ clone at that time-point. Mean ± SE and ANOVA <span class="html-italic">p</span>-values (n = 7) for effects of three factors (water availability, genotype, and day of observation) and their interactions are shown. The significant <span class="html-italic">p</span>-values were marked in bold.</p>
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<p>Representative area of leaf xylem vessel (µm<sup>2</sup>) measured in <span class="html-italic">C. canephora</span> var. Robusta clones (‘3V’ and ‘A1’) under well-watered (WW) and water stress (WS) conditions: (<b>A</b>) A1-WW, (<b>B</b>) 3V-WW, (<b>C</b>) A1-WS, and (<b>D</b>) 3V-WS, evaluated at the end of the second drought cycle. A scale of 100 µm is shown.</p>
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<p>Diagram of the two drought cycles. Transplant followed by drought-1 event last for 19 days (until −500 kPa of Ψ<sub>msoil</sub> was reached), followed by a 31-day period for a whole plant recovery (including 12-day period of recovery-1 event). The 2nd drought cycle was then applied, similarly to the 1st drought cycle, by withholding irrigation until the −500 kPa of Ψ<sub>msoil</sub> was reached (drought-2 event) and followed by another 12 days of recovery-2 event.</p>
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17 pages, 4394 KiB  
Review
Vascular NADPH Oxidases and Atherothrombotic Stroke
by Javier Marqués and Guillermo Zalba
Stresses 2024, 4(3), 558-574; https://doi.org/10.3390/stresses4030036 - 5 Sep 2024
Viewed by 253
Abstract
Oxidative stress constitutes a main molecular mechanism underlying cardiovascular diseases (CVDs). This pathological mechanism can be triggered by NADPH oxidases (NOXs), which produce reactive oxygen species (ROS). In fact, the different NOXs have been associated with myocardial infarction, atherothrombosis, and stroke. More specifically, [...] Read more.
Oxidative stress constitutes a main molecular mechanism underlying cardiovascular diseases (CVDs). This pathological mechanism can be triggered by NADPH oxidases (NOXs), which produce reactive oxygen species (ROS). In fact, the different NOXs have been associated with myocardial infarction, atherothrombosis, and stroke. More specifically, we will focus on the implications of NOXs in atherothrombotic stroke. Each NOX member participates in a different way in the several stages of this disease: endothelial dysfunction, immune cell infiltration, foam cell genesis, vascular smooth muscle cells (VSMC) proliferation, and atherosclerotic plaque formation. Additionally, some NOXs are involved in plaque instability, thrombosis, ischemic stroke, and ischemia-reperfusion injury (IRI). Interestingly, the effects of NOXs in this pathology depend on the specific homolog, the cell type in which they are activated, and the stage of the disease. In this review we summarize the most up-to-date information about the implications of vascular NOXs in each of these processes. Finally, we highlight some limitations and future perspectives on the study of NOXs in CVDs. Full article
(This article belongs to the Collection Feature Papers in Human and Animal Stresses)
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Figure 1

Figure 1
<p>Intracellular distribution of NOX homologs in endothelial cells, VSMC, and monocytes/macrophages. Endothelial Cells. NOX1 is expressed, but its intracellular localization remains unknown. NOX2 is located in membrane protrusions and in association with the cytoskeleton. NOX4 and NOX5 are located in the endoplasmic reticulum. <b>Vascular Smooth Muscle Cells</b>. NOX1 is located inside clathrin-coated areas of the membrane when inactive and translocases to the membrane upon activation. NOX2 is expressed, but its intracellular location remains unknown. NOX4 is located inside the mitochondria, at the nucleus, and at focal adhesions. NOX5 is located in cholesterol-rich areas of the plasma membrane. <b>Monocytes/Macrophages.</b> NOX1 is expressed, but its intracellular localization remains unknown. NOX2 is located at intracellular membranes and translocases to the plasma membrane upon activation. NOX4 is located at the endoplasmic reticulum and the nucleus. NOX5 is located in mitochondria.</p>
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<p>NOX overactivation leads to endothelial cell dysfunction and inflammation. Humoral factors related to atherosclerosis (Ang II, ET-1, oxLDL) increase NOX activity. The overactivation of every NOX leads to apoptosis. The activation of NOX2, NOX4, and NOX5 by TLRs ends in cytokines secretion. NOX4 and NOX5 alter PG signaling, inflammatory mediators that participate in atherosclerosis. Ang II: angiotensin II. ET-1: endothelin 1. GM-CSF: granulocyte-monocyte colony-stimulating factor. MCP-1: monocyte chemoattractant protein 1. oxLDL: oxidized LDL. PGE<sub>2</sub>: prostaglandin E<sub>2</sub>. PGI<sub>2</sub>: prostaglandin I<sub>2</sub>. TLR: toll-like receptor.</p>
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<p>The different NOX homologs play crucial roles in the immune cell infiltration into the vascular wall and the genesis of foam cells. NOX2, NOX4, and NOX5 increase ICAM-1 and VCAM-1 expression in endothelial cells, promoting monocyte adhesion. By contrast, NOX4 inhibits the infiltration of monocytes. NOX2 promotes the apoptosis of macrophages which aggravates the immune cell infiltration by inflammatory signals. NOX1 mediates the transformation of macrophages towards foam cells. NOX2 activation leads to alterations in ECM composition, cell migration, and lipid accumulation in VSMC. NOX1, NOX4 and NOX5 also participate in VSMC migration. ECM: extracellular matrix. ICAM-1: intracellular adhesion molecule 1. LPS: lipopolysaccharide. TNF-α: tumoral necrosis factor α. VCAM-1: vascular cell adhesion molecule 1.</p>
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<p>The NOX homologues play crucial roles in atherosclerotic plaque growth and thrombosis. NOX1 and NOX2 increase VSMC proliferation, while the role of NOX4 in this step is controversial. NOX2 and NOX5 promote VSMC switch toward a synthetic phenotype and Ca<sup>++</sup> accumulation. NOX1, NOX2, and NOX4 promote plaque progression in vivo, while NOX5 inhibits this process in rabbits. NOX1 and NOX2 are prothrombotic, while NOX4 has no effect in this process.</p>
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12 pages, 1498 KiB  
Article
Enhanced Production of Therapeutic Metabolites in Cork-Oak Somatic Embryos under Abiotic Stress Conditions
by Beatriz Pintos López, José Antonio Manzanera, Elena Pérez-Urria, Carlos Jiménez, Alba Montoro and Arancha Gomez-Garay
Stresses 2024, 4(3), 546-557; https://doi.org/10.3390/stresses4030035 - 3 Sep 2024
Viewed by 397
Abstract
This study investigates the enhanced production of key therapeutic metabolites (ellagic acid, serotonin, and chlorogenic acid) in response to abiotic stress in in vitro cultures of Quercus suber somatic embryos. Findings indicate significant increases in metabolite levels under various stress conditions, highlighting the [...] Read more.
This study investigates the enhanced production of key therapeutic metabolites (ellagic acid, serotonin, and chlorogenic acid) in response to abiotic stress in in vitro cultures of Quercus suber somatic embryos. Findings indicate significant increases in metabolite levels under various stress conditions, highlighting the potential for commercial-scale production of these compounds, known for their antioxidant, anticancer, and anti-COVID-19 properties. Under osmotic/saline stress, ellagic acid production significantly increased, representing an 80% increase compared to control conditions. In embryos exposed to different stressors, serotonin accumulation showed a six-fold increase under osmotic/saline stress. Although the elicitors used did not increase chlorogenic acid levels, exploring alternative stress types may enhance its production. This research paves the way for sustainable, large-scale production of health-beneficial metabolites, addressing global health challenges and promoting resource sustainability. Full article
(This article belongs to the Section Plant and Photoautotrophic Stresses)
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Figure 1

Figure 1
<p>(<b>a</b>) Direct somatic embryogenesis on an immature zygotic embryo after 30 days in SM medium. (<b>b</b>) Embryos and embryonic callus on SM medium supplemented with 500 mg/L Glutamine, showing recurrent embryogenesis and numerous dicotyledonary embryos.</p>
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<p>Plant polyphenols: Ellagic acid (<b>a</b>) and Chlorogenic acid (<b>b</b>) content (mg/kg dry weight, DW) measured by LC-QQQ-MS in <span class="html-italic">Quercus suber</span> somatic embryos subjected to different abiotic stress treatments: high temperature (35 °C), UV radiation, moderate osmotic/saline stress (50 mM NaCl), severe osmotic/saline stress (200 mM NaCl), and cerium oxide nanoparticles (nanoceria) at concentrations of 100 mg/L and 200 mg/L in the culture medium. Treatments sharing the same letter were not statistically significant at the 0.05 level, as determined by Duncan’s test. The inset on the right shows the chromatograms of Ellagic acid detection in control sample under two different collision energies (CE: −41.0, black, and −33.0, pink), and Chlorogenic acid (CE: −17.0, blue, and −47.0, red).</p>
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<p>Serotonin content (mg/kg dry weight, DW) measured by LC-QQ-MS in <span class="html-italic">Quercus suber</span> somatic embryos subjected to different abiotic stress treatments: high temperature (35 °C), UV radiation, moderate osmotic/saline stress (50 mM NaCl), severe osmotic/saline stress (200 mM NaCl), and cerium oxide nanoparticles (nanoceria) at concentrations of 100 mg/L and 200 mg/L in the culture medium. Treatments sharing the same letter were not statistically significant at the 0.05 level, as determined by Duncan’s test. The inset on the right shows the chromatograms of serotonin detection in control sample under two different collision energies (CE: −13.0 and −29.0), highlighting the transition from <span class="html-italic">m</span>/<span class="html-italic">z</span> 176.60 to 160.00 (black) and <span class="html-italic">m</span>/<span class="html-italic">z</span> 176.60 to 115.00 (pink).</p>
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12 pages, 1729 KiB  
Article
Dynamics of Stress Biomarker in Cliff Divers during Official Competition
by Mia Perić, Vladimir Pavlinović, Nikola Foretić, Zoran Nikolovski and Dario Vrdoljak
Stresses 2024, 4(3), 534-545; https://doi.org/10.3390/stresses4030034 - 23 Aug 2024
Viewed by 299
Abstract
Stress is defined as a reaction of the body to any given stressor, external or internal. These stressors are common among participants in sports. Therefore, this study aimed to determine the dynamics of two stress biomarkers during an official cliff diving competition. The [...] Read more.
Stress is defined as a reaction of the body to any given stressor, external or internal. These stressors are common among participants in sports. Therefore, this study aimed to determine the dynamics of two stress biomarkers during an official cliff diving competition. The sample of participants included six professional cliff divers (three females and three males). Their salivary cortisol (C) and alpha-amylase (AA) samples were collected during a 3-day competition (four samples on day 1 and six samples on days 2 and 3). The analysis of the results showed a non-significant increase in AA from day one to day three. On the other hand, C has an increase in the first two days (0.58 ± 0.16 µg/mL–0.61 ± 0.23 µg/mL) and then a decrease in the last competition day (0.53 ± 0.23 µg/mL). Analysis of samples collected during each day demonstrates a fluctuation of the biomarkers. For AA, the sample after the training dive on the 2nd day has the highest value (326.34 ± 280.73 U/mL), whereas on day 3, the samples after training and before the competition dive are the highest (364.50 ± 287.13 U/mL; 466.49 ± 218.39 U/mL). Regarding C levels, the sample after the competition dive tends to show the highest values (0.66 ± 0.17 µg/mL; 0.89 ± 0.29 µg/mL; 0.76 ± 0.32 µg/mL, respectively). Furthermore, the correlation between the results of competition and biomarkers is not significant. These results demonstrate how cliff diving competition tends to have a high impact on the sympathetic nervous system, as observed mainly in AA dynamics. One might contend that even though there is no significant physical strain, cliff divers demonstrate heightened stress biomarker levels that could affect their performance and focus while diving. Full article
(This article belongs to the Collection Feature Papers in Human and Animal Stresses)
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Figure 1
<p>Post-hoc analysis with arithmetic means and standard deviations for AA (black) and C (grey) among days of the competition with the number of samples (N).</p>
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<p>T-test analysis with arithmetic means and standard deviations for AA (black) and C (grey) between training and competition dives with the number of samples (N).</p>
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<p>Correlation analysis between C and AA with grade of dive during competition for before dive AA (<b>A</b>), after dive AA (<b>B</b>), before dive C (<b>C</b>), and after dive C (<b>D</b>). (dots) representing individual values; (black line) linear regression plot; (shade areas) 95% confidence interval.</p>
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<p>T-test analysis with arithmetic means and standard deviations between males and females in AA (<b>black</b>) and C (<b>grey</b>).</p>
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<p>Correlation analysis between C and AA during day 1 (<b>A</b>), day 2 (<b>B</b>), and day 3 (<b>C</b>). (dots) representing individual values; (black line) linear regression plot; (shade areas) 95% confidence interval.</p>
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<p>Sample collection timeline with mean sample data before the dive (training and competition) and directly after the dive (training and competition)—some sample times differ from the timeline due to the different duration of dives.</p>
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16 pages, 3672 KiB  
Article
Genome-Wide Identification, Bioinformatic Characterization, and Expression Profiling of Starch Synthase (SS) Genes in Foxtail Millet under Drought Condition
by Joseph N. Amoah, Monica Ode Adu-Gyamfi and Albert Owusu Kwarteng
Stresses 2024, 4(3), 518-533; https://doi.org/10.3390/stresses4030033 - 16 Aug 2024
Viewed by 546
Abstract
Millet, a vital and nutritionally dense cereal extensively cultivated in Sub-Saharan Africa, plays a key role in ensuring food security. This study investigates the starch synthase (SS) gene family, which is crucial for starch biosynthesis and influences various plant functions and [...] Read more.
Millet, a vital and nutritionally dense cereal extensively cultivated in Sub-Saharan Africa, plays a key role in ensuring food security. This study investigates the starch synthase (SS) gene family, which is crucial for starch biosynthesis and influences various plant functions and stress responses. While the specific roles of SS genes in millet under drought conditions are not fully elucidated, this research provides a thorough analysis of the SS gene family in millet. A total of twelve millet SS genes (SiSSs) were identified and classified into four subfamilies (I–IV) through gene structure and phylogenetic analysis. The SiSS genes were unevenly distributed across millet chromosomes, with cis-acting elements associated with plant growth and stress defense being identified. Quantitative PCR (qPCR) revealed dynamic and varied expression patterns of SiSSs in different tissues under drought stress. Millet plants subjected to drought conditions showed higher tissue starch content and increased starch synthase activity compared to controls. Importantly, the expression levels of the twelve SiSSs were positively correlated with both starch content and synthase activity, suggesting their significant role in drought tolerance. This study enhances our understanding of the millet SS gene family and highlights the potential of these genes in breeding programs aimed at developing drought-resistant millet varieties. Further research is recommended to validate these findings and delve deeper into the mechanisms underlying drought tolerance. Full article
(This article belongs to the Topic Plant Responses to Environmental Stress)
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Figure 1
<p>The phylogenetic relationships among SS proteins in five plant species are depicted through a phylogenetic tree. SS proteins from millet (SiSS1-SiSS12), Arabidopsis thaliana (AtSS1-AtSS7), wheat (TaSS1-TaSS7), rice (OsSS1-OsSS14), and cassava (MeSS1-MeSS4) are highlighted in red, blue, yellow, pink, and cyan-blue colors, respectively. The neighbor-joining phylogenetic tree of SS protein sequences was constructed with 1000 bootstrap replicates using MEGA v10.0 (Pennsylvania State University, Philadelphia, PA, USA).</p>
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<p>(<b>A</b>) Gene structure of <span class="html-italic">SiSS</span> genes generated using the GSDS 2.0 (Gene Structure Display Server) website (<a href="http://gsds.cbi.pku.edu.cn" target="_blank">http://gsds.cbi.pku.edu.cn</a> (accessed on 20 March 2024)). (<b>B</b>) Exon numbers of p-tative <span class="html-italic">SiSSs</span>. (<b>C</b>) Conserved motifs of the <span class="html-italic">SiSSs</span> identified by MEME (Multiple EM for Motif Elicitation). Each motif is represented by a colored box numbered at the bottom, and the length of the motifs in each protein is proportional. The phylogenetic tree was constructed using the maximum likelihood method with 1000 bootstrap replicates by MEGA v10.0 (Pennsylvania State University, Philadelphia, PA, USA).</p>
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<p>(<b>A</b>) Distribution of <span class="html-italic">SiSSs</span> on the millet chromosome, and (<b>B</b>) gene duplication analysis of <span class="html-italic">SiSSs</span>. The putative whole genome duplicated (WGD) genes are connected by various lines. Numbers 1-9 indicate the positions of SSs on chromosomes 1 through 9 in millet.</p>
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<p>Synteny analysis of <span class="html-italic">SiSSs</span> among millet, Arabidopsis, rice, tomato, and barley was conducted using the one-step MCScanX on TBtools for gene duplication analysis. Grey lines in the background represent collinear blocks within the genomes of different plant species, and blue lines indicate syntenic SS gene pairs. Numbers 1-9 indicate the positions of <span class="html-italic">SSs</span> on chromosomes 1 through 9 across various plant species, while the triangles mark the exact locations of the <span class="html-italic">SSs</span> genes on each chromosome.</p>
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<p>Cis-acting regulatory element (CARE) analysis identified in the putative starch synthesis genes. The CAREs were analyzed from the upstream (2000 bp) promoter sequence of each gene. Identified CAREs were classified based on their function into (<b>A</b>) growth and development, (<b>B</b>) phytohormones, (<b>C</b>) stress and defense, and (<b>D</b>) light-responsive elements.</p>
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<p>Three-dimensional (3D) modeling of starch synthase (<span class="html-italic">SS</span>) Models were predicted and displayed at a confidence level &gt; 90%. Green and blue helix structures denote alpha helix (%) and beta strand (%), respectively.</p>
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<p>Transmembrane topology prediction of <span class="html-italic">SS</span> proteins in millet. Models were predicted and displayed at a confidence level &gt; 90%.</p>
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<p>Quantitative polymerase chain reaction (qPCR) expression analysis of putative <span class="html-italic">SiSSs</span> under different drought conditions. (<b>A</b>–<b>L</b>) represent the relative tissue expression (leaf and root) of putative <span class="html-italic">SiSSs</span> (<span class="html-italic">SiSS1</span>-<span class="html-italic">SiSS12</span>) after a 15-day drought stress treatment. The data represent the mean and standard errors of biological triplicates. Significance (<span class="html-italic">p</span> &lt; 0.05, <span class="html-italic">p</span> &lt; 0.001) was determined using <span class="html-italic">t</span>-test.</p>
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<p>Heat map depicting the expression profiles of <span class="html-italic">SiSS</span> genes after a 15-day drought treatment in (<b>A</b>) leaf and (<b>B</b>) root tissues of the ‘PI 662292’ millet genotype. The heat map was computed using the mean expression values of each putative gene. I–III denotes the three different groups of classification for <span class="html-italic">SiSSs</span>.</p>
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13 pages, 10879 KiB  
Article
Rosmarinic Acid Attenuates Testicular Damage via Modulating Oxidative Stress and Apoptosis in Streptozotocin-Induced Diabetic Albino Mice
by Omar Al-khawaldeh, Zina M. Al-Alami, Osama Y. Althunibat, Tamer M. M. Abuamara, Afnan Mihdawi and Mohammad H. Abukhalil
Stresses 2024, 4(3), 505-517; https://doi.org/10.3390/stresses4030032 - 5 Aug 2024
Viewed by 647
Abstract
Diabetes mellitus (DM) induces the production of reactive oxygen species, which may lead to cell injury and death. This study aimed to assess the effects of rosmarinic acid (RA) on testicular damage, oxidative stress, and apoptosis in streptozotocin (STZ)-induced diabetic albino mice. DM [...] Read more.
Diabetes mellitus (DM) induces the production of reactive oxygen species, which may lead to cell injury and death. This study aimed to assess the effects of rosmarinic acid (RA) on testicular damage, oxidative stress, and apoptosis in streptozotocin (STZ)-induced diabetic albino mice. DM in four- to six-week-old BALB/c male albino mice was induced via 50 mg/kg STZ, IP for 5 days. Twelve mice were randomly assigned into each of following groups: a control group, a diabetic (DM) group, RA5 mg/kg and RA15 mg/kg groups, and DM + RA5 mg/kg and DM + RA15 mg/kg groups. RA doses were intraperitoneally injected six times a week for seven weeks. Diabetes increased blood sugar and HbA1c levels and decreased all assessed sperm parameters. Testicular tissues of the diabetic mice showed increased lipid peroxidation, decreased reduced glutathione levels and catalase and superoxide dismutase activities, and increased apoptosis associated with histological abnormalities. Both RA doses had no effects on final body weight, blood sugar, and HbA1c in the diabetic mice. It is concluded that the administration of the potent antioxidant RA to diabetic mice improved the redox status in testicular tissues, protected them from diabetes-induced oxidative damage, and improved the quality of spermatozoa, mostly in a dose-dependent manner, which suggests a potential application value of RA in treating DM-related testicular injury and perhaps other complications. Full article
(This article belongs to the Section Animal and Human Stresses)
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<p>Effects of RA on body weight, FBG levels, and HbA1c in diabetic mice. (<b>a</b>) Average mean body weight during the experimental period. (<b>b</b>) Average mean values of fasting glucose level before and during treatment. (<b>c</b>) HbA1c levels. All values are expressed as the mean ± SEM. The letters on the bars mean the following: a: significant difference when compared to the control group at <span class="html-italic">p</span> &lt; 0.05; b: significant difference when compared to the DM group at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Effects of RA on sperm parameters in diabetic mice: (<b>a</b>) Sperm count. (<b>b</b>) Percentage of motile sperms. (<b>c</b>) Percentage of viable sperm. (<b>d</b>) Percentage of sperm with normal morphology. All values are expressed as the mean ± SEM. The letters above the bars mean the following: a: significant difference when compared to the control group at <span class="html-italic">p</span> &lt; 0.05; b: significant difference when compared to the DM group at <span class="html-italic">p</span> &lt; 0.05; ab: significant difference when compared to the control and DM groups at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Effects of RA on oxidative stress in the testis of diabetic mice: (<b>a</b>) TBARS levels. (<b>b</b>) Concentration of reduced glutathione (GSH). (<b>c</b>) SOD activity. (<b>d</b>) Catalase (CAT) absorbance. All values are expressed as the mean ± SEM. The letters above the bars mean the following: a: significant difference when compared to the control group at <span class="html-italic">p</span> &lt; 0.05; b: significant difference when compared to the DM group at <span class="html-italic">p</span> &lt; 0.05; ab: significant difference when compared to control and DM group at <span class="html-italic">p</span> &lt; 0.05.</p>
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<p>Photomicrographs obtained from the testes of mice from (<b>a</b>) the control group, (<b>b</b>) the DM group, (<b>c</b>) the RA5 mg/kg group, (<b>d</b>) the RA15 mg/kg group, (<b>e</b>) the DM + RA5 mg/kg group, and (<b>f</b>) the DM + RA15 mg/kg group (X400; H&amp;E); scale bar: 100 uM. Early spermatids (ES), fibroblasts (F), hyalinization in the lumen of some seminiferous tubules (H), interstitial cells of Leydig (LC), late spermatids (LS), myoid cells (M), primary spermatocytes (PS), pyknotic and necrotic spermatogenic cells (Pk), seminiferous tubules (ST), Sertoli cells (SC), spermatid giant cells (GC), spermatids (S), spermatogonia (SG), vacuoles (V). Sloughing germinal epithelium and irregular basement membrane (*), absence of spermatogenesis (red circle).</p>
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<p>Representative images of TUNEL-stained sections in the testes of mice: (<b>a</b>) Control group. (<b>b</b>) DM group. (<b>c</b>) RA5 mg/kg group. (<b>d</b>) RA15 mg/kg group. (<b>e</b>) DM + RA5 mg/kg group. (<b>f</b>) DM + RA15 mg/kg group. In these fluorescent microscopic images, the bright green color represents DNA-fragmented cells, and the blue color represents the DAPI stain, meaning intact DNA. Scale bar: 10 μm.</p>
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24 pages, 1732 KiB  
Review
Endoplasmic Reticulum Stress Signaling in the Regulation of Hepatic Pathological Responses
by Ananda Baral
Stresses 2024, 4(3), 481-504; https://doi.org/10.3390/stresses4030031 - 1 Aug 2024
Viewed by 706
Abstract
The endoplasmic reticulum (ER) is a vital cell organelle that is primarily involved in the processes of protein folding, maintenance of intracellular calcium storage and lipid synthesis in order to maintain cellular homeostasis. To achieve this meticulous order, several ER-dependent processes have to [...] Read more.
The endoplasmic reticulum (ER) is a vital cell organelle that is primarily involved in the processes of protein folding, maintenance of intracellular calcium storage and lipid synthesis in order to maintain cellular homeostasis. To achieve this meticulous order, several ER-dependent processes have to be in unison and perfect harmony. However, a persistent supply of newly synthesized proteins strains the ER mainly due to the accumulation of unfolded proteins, thus ultimately leading to an imbalance termed ER stress. Although the accumulation of misfolded proteins is a frequent reason for the initiation of ER stress, it is also induced by the hyper-production of reactive oxygen species, aberrant calcium leakage from the ER and due to the effect of cytokines. ER stress signals are conveyed via three arms of ER, namely PERK, IRE1 and ATF6. Signal transduction form these signaling molecules often converges on the transcriptional upregulation of CHOP and its related signaling mechanisms. If the ER stress is unresolved, then it can lead to cell death through different cell death mechanisms, including apoptosis, proptosis, etc. In the liver, it has been observed that ER stress plays a critical role in hepatic damage under different experimental conditions. This review highlights the role of ER stress in liver pathologies. Full article
(This article belongs to the Collection Feature Papers in Human and Animal Stresses)
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<p>Multipotent role of ER stress in liver disease. ER stress is known to influence a wide variety of liver diseases that can range from simple steatosis, which arises because of excess fat deposition in hepatocytes, to severe pathologies such as HCC. The major causative role of ER stress in promoting liver pathologies lies in the abnormal activation of immune responses and metabolic dysfunction. This illustration was partially created with BioRender.com.</p>
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<p>Branches of UPR and their signaling response. UPR transduces signal downstream via three transmembrane domains of the ER. This occurs subsequent to the release of BiP, resulting in the transautophosphorylation of PERK and IRE1α and liberation of ATF6α from ER followed by translocation into Golgi. All of these events generate bZIP transcription factors, which are implicated in the gene expression of various molecules, including ER chaperones and proteins associated with ERAD, autophagy and apoptosis.</p>
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<p>Cellular events that modulate cell fate under ER stress. ER stress can influence both the cytoprotective/adaptive and cytotoxic responses. This decision is often associated with the strength and time duration of ER stress inducing stimuli. As ER stress can also promote the activation of chaperone proteins and some antioxidant genes, the expression levels of these proteins also modulate cell fate. Upward facing arrow indicates upregulation and downward facing arrow indicates downregulation of respective system.</p>
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<p>The many facets of ER stress. ER stress can influence various sets of mechanisms that can include upregulation in ROS, the release of calcium into the cytosol, promote inflammasome activation, and induce hepatic steatosis, thereby modulating hepatic physiology in a variety of ways and often leading to austere consequences. Here the terminology “facets” is supposed to be understood as the diverse signaling mechanisms that are modulated by ER stress. These events can be lipid accumulation, ROS production, calcium release and upregulation of cytokines. In recent years, the connection between ER stress and inflammasome activation has received due attention. Reciprocally, these mechanisms also induce ER stress.</p>
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29 pages, 390 KiB  
Review
Strategic Advancements in Rice Cultivation: Combating Heat Stress through Genetic Innovation and Sustainable Practices—A Review
by Pretty Mthiyane, Murat Aycan and Toshiaki Mitsui
Stresses 2024, 4(3), 452-480; https://doi.org/10.3390/stresses4030030 - 25 Jul 2024
Viewed by 564
Abstract
Rice is one of the most important staple foods globally, sustaining over half of the world’s population. However, the sustainability of grain production is increasingly threatened by heat stress, which is intensified by global climate change. Heat stress, characterized by temperatures exceeding crop-specific [...] Read more.
Rice is one of the most important staple foods globally, sustaining over half of the world’s population. However, the sustainability of grain production is increasingly threatened by heat stress, which is intensified by global climate change. Heat stress, characterized by temperatures exceeding crop-specific optimal growth thresholds, significantly impacts the rice yield and quality, particularly during critical reproductive stages. This review synthesizes current research on strategies to mitigate heat stress in rice through genetic and agronomic approaches. It highlights the implementation of advanced genetic tools such as marker-assisted selection (MAS) and genomic selection (GS) to accelerate the breeding of heat-tolerant rice varieties. Additionally, it discusses sustainable agronomic practices, including adjusting planting dates, optimizing water management, and crop rotation, which enhance resilience to heat stress. The objective of this review is to bridge the gap between research findings and practical agricultural applications, providing a comprehensive resource that guides future research directions and informs policy interventions. This review emphasizes the importance of integrating genetic innovations with traditional and modern farming practices to develop rice varieties that can withstand the adverse effects of heat stress, ensuring food security and agricultural sustainability in the face of climatic challenges. Full article
16 pages, 2567 KiB  
Article
Environmental Cadmium Exposure Induces an Increase in Systolic Blood Pressure by Its Effect on GFR
by Soisungwan Satarug, David A. Vesey, Supabhorn Yimthiang, Tanaporn Khamphaya, Phisit Pouyfung and Aleksandra Buha Đorđević
Stresses 2024, 4(3), 436-451; https://doi.org/10.3390/stresses4030029 - 15 Jul 2024
Viewed by 802
Abstract
Chronic exposure to the nephrotoxic metal pollutant, cadmium (Cd), has been associated with hypertension, but the mechanism by which it raises blood pressure is not understood. We hypothesize that exposure to Cd reduces the glomerular filtration rate (GFR), which in turn causes a [...] Read more.
Chronic exposure to the nephrotoxic metal pollutant, cadmium (Cd), has been associated with hypertension, but the mechanism by which it raises blood pressure is not understood. We hypothesize that exposure to Cd reduces the glomerular filtration rate (GFR), which in turn causes a rise in blood pressure. Data were collected from 447 Thai subjects with a mean age of 51.1 years, of which 48.8% had hypertension, 15.4% had diabetes, and 6.9% had an estimated GFR (eGFR) below 60 mL/min/1.73 m2 (low eGFR). More than half (58.8%) and 23.9% had moderate and severe tubular proteinuria, respectively. The mean blood and urinary Cd concentrations were 2.75 and 4.23 µg/L, respectively. Doubling of body burden of Cd increased the prevalence odds ratios (POR) for low eGFR and severe tubular proteinuria 41% and 48%, respectively. The POR for hypertension rose twofold in those with blood Cd levels of 0.61–1.69 µg/L or urinary Cd excretion levels ≥ 0.98 µg/g creatinine. In the hypertensive group, the eGFR was inversely associated with age (β = −0.517), the Cd excretion rate (β = −0.177), and diabetes (β = −0.175). By mediation analysis, an increase in SBP was attributable totally to the effect of Cd on GFR. Thus, blood pressure appeared to rise as GFR fell. This finding is consistent with the well-known role of the kidney in long-term blood pressure regulation, and explains a universally high prevalence of hypertension among patients with low eGFR. Full article
(This article belongs to the Collection Feature Papers in Human and Animal Stresses)
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<p>Comparing cadmium effects on GFR in women and men. Scatterplots relate eGFR to log[(E<sub>Cd</sub>/C<sub>cr</sub>) × 10<sup>5</sup>] in women and men with (E<sub>Cd</sub>/C<sub>cr</sub>) × 100 &lt; 1 µg/L filtrate (<b>a</b>) and E<sub>Cd</sub>/C<sub>cr</sub>) × 100 ≥ 1 µg/L filtrate (<b>b</b>). Coefficients of determination (R<sup>2</sup>) and standardized β-coefficients for all scatterplots, numbers of subjects in subgroups, and <span class="html-italic">p</span>-values are provided.</p>
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<p>Comparing the effects of cadmium on GFR in the normotensive and hypertensive groups. Scatterplots relate eGFR to log[(ECd/Ccr) × 105] in normotensive and hypertensive with (E<sub>Cd</sub>/C<sub>cr</sub>) × 100 &lt; 1 µg/L filtrate (<b>a</b>) and E<sub>Cd</sub>/C<sub>cr</sub>) × 100 ≥ 1 µg/L filtrate (<b>b</b>). Coefficients of determination (R2) and standardized β-coefficients for all scatterplots, numbers of subjects in subgroups, and <span class="html-italic">p</span>-values are provided.</p>
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<p>Cadmium and eGFR as predictors of blood pressure increases. Scatterplots relate SBP (<b>a</b>,<b>b</b>) and DBP (<b>c</b>,<b>d</b>) to eGFR in women and men with (E<sub>Cd</sub>/C<sub>cr</sub>) × 100 of &lt;1 and ≥ 1 µg/L filtrate. Coefficients of determination (R<sup>2</sup>) and standardized β-coefficients for all scatterplots, numbers of participants in subgroups, and <span class="html-italic">p</span>-values are provided.</p>
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<p>Mediation analysis of the effect of cadmium on blood pressure in the high-exposure group. (<b>a</b>) A model depicts eGFR as a mediator of the effect of Cd on blood pressure increases and standardized β values (<b>b</b>) The Sobel test of unstandardized β coefficients describing relationships of Cd with eGFR (A), eGFR with blood pressure (B), and Cd with blood pressure (C′).</p>
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<p>Mediation analysis of the effect of cadmium on blood pressure in the low-exposure group. (<b>a</b>) A model depicts eGFR as a mediator of the effect of Cd on blood pressure increases and standardized β values. (<b>b</b>) The Sobel test of unstandardized β coefficients describing relationships of Cd with eGFR (A), eGFR with blood pressure (B), and Cd with blood pressure (C′).</p>
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15 pages, 516 KiB  
Article
Silica Wort Supplementation as an Alternative for Yeast Stress Relief on Corn Ethanol Production with Cell Recycling
by Matheus Ribeiro Barbosa Oliveira, Rafael Soares Douradinho, Pietro Sica, Layna Amorim Mota, Alana Uchôa Pinto, Tamires Marques Faria and Antonio Sampaio Baptista
Stresses 2024, 4(3), 421-435; https://doi.org/10.3390/stresses4030028 - 8 Jul 2024
Viewed by 478
Abstract
In very high gravity (VHG) fermentation, yeast cells are subjected to a multitude of challenging conditions, including the osmotic pressure exerted by the high sugar content of the wort and the stress factors associated with the high ethanol concentrations present at the end [...] Read more.
In very high gravity (VHG) fermentation, yeast cells are subjected to a multitude of challenging conditions, including the osmotic pressure exerted by the high sugar content of the wort and the stress factors associated with the high ethanol concentrations present at the end of the fermentation cycle. The response of this biological system to abiotic stresses may be enhanced through biochemical and physiological routes. Silica may play a significant role in regulating the cellular homeostasis of yeast. Alternatively, it is expected that this outcome may be achieved through biochemical responses from the effects of vitamins on yeast cells and the physiological yeast route changing by the culture medium aeration. The objective of this study was to investigate the effects of adding 500 mg L−1 of silica on corn ethanol wort medium and the possibility of supplementing the same wort with vitamins alongside aeration (0.2 v v−1 min−1) as an alternative resource to sustain the fermentation yield rather than adding silica in a fed-batch fermentation cycle with yeast recycling. Upon completion of the five fermentation cycles, yeast samples subjected to the treatment with the addition of silica exhibited a 3.1% higher fermentation yield in comparison to the results observed in the vitamins plus aeration medium bath. Even though greater biomass production (19.1 g L−1) was observed through aerobic yeast behavior in vitaminized supplemented corn medium, the provided silica had a more beneficial effect on yeast stress relief for very high gravity fermentation in a corn hydrolyzed wort with cell recycling. Full article
(This article belongs to the Collection Feature Papers in Plant and Photoautotrophic Stresses)
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<p>Illustration of the fermentative assay in the present study.</p>
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10 pages, 2685 KiB  
Communication
Cell-Type-Specific Effect of Innate Immune Signaling on Stress Granules
by Prem Prasad Lamichhane, Aditi, Xuping Xie and Parimal Samir
Stresses 2024, 4(3), 411-420; https://doi.org/10.3390/stresses4030027 - 5 Jul 2024
Viewed by 830
Abstract
Stress granules (SGs) are cytoplasmic membraneless compartments that can form in stressed cells. There is an intricate relationship between SGs and innate immune signaling pathways. A previous study reported that the innate immune signaling mediated by Toll-like receptors (TLRs) can inhibit SGs induced [...] Read more.
Stress granules (SGs) are cytoplasmic membraneless compartments that can form in stressed cells. There is an intricate relationship between SGs and innate immune signaling pathways. A previous study reported that the innate immune signaling mediated by Toll-like receptors (TLRs) can inhibit SGs induced by endoplasmic reticulum stress (ER stress) in bone-marrow-derived macrophages (BMDMs) and the chemotherapy drug oxaliplatin in B16 melanoma cells. We wanted to test if this observation can be generalized to other cell types. First, we recapitulated the results from the previous study showing TLR signaling-mediated inhibition of SGs in BMDMs induced by ER stress. However, SGs formed in response to ER stress were either not inhibited or only very weakly inhibited by TLR4 stimulation in human lung cancer-derived A549 cells, murine immortalized mouse lung fibroblasts (iMLFs) and primary murine mouse lung fibroblasts. This correlated with a weak induction of IKK complex kinase activity by TLR4 stimulation in these cells. SGs formed by sodium arsenite treatment also remained unaffected by TLR4 signaling. Our results indicate that the innate immune signaling-mediated inhibition of SGs is cell-type-dependent, thus opening a new avenue for mechanistic studies of the crosstalk between innate immune and stress signaling pathways. Full article
(This article belongs to the Collection Feature Papers in Human and Animal Stresses)
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<p>TLR signaling inhibits ER stress-induced SGs in BMDMs. (<b>A</b>) Effect of TLR4 stimulation using 100 ng/mL LPS for 6 h on ER stress-induced SGs. (<b>B</b>) Effect of TLR7 stimulation using 1 µg/mL R848 for 6 h on ER stress-induced SGs. (<b>C</b>) Effect of TLR2 stimulation using 1 µg/mL Pam3CSK4 for 6 h on ER stress-induced SGs. (<b>D</b>) Effect of TLR3 stimulation using 50 µg/mL poly(I:C) for 6 h on ER stress-induced SGs. Abbreviations: pI:C = poly(I:C), 2 µg/mL Thap = thapsigargin for 90 min, Pam3 = Pam3CSK4. DAPI was used to mark nuclei. **** denotes <span class="html-italic">p</span> &lt; 0.0001 using the two-tailed Mann–Whitney test.</p>
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<p>TLR signaling only weakly inhibits ER stress-induced SGs in A549 cells. (<b>A</b>) Effect of TLR4 stimulation using 1 µg/mL LPS for 6 h on ER stress- and sodium arsenite-induced SGs. (<b>B</b>) Quantification of SG assembly induced by ER stress. (<b>C</b>) Quantification of SG assembly induced by sodium arsenite. Abbreviations: Thap = 1 µg/mL thapsigargin for 60 min, Ars = 500 µM sodium arsenite for 60 min. <span style="color:blue">Blue: DAPI</span>, <span style="color:lime">green: G3BP1</span>, <span style="color:red">red: DDX3X</span>. ** denotes <span class="html-italic">p</span> &lt; 0.01 using the two-tailed Mann–Whitney test. (<b>D</b>) Effect of translation inhibition on arsenite-induced SG assembly in A549 cells. Cycloheximide was used to stabilize polysomes and inhibit SG assembly. Puromycin did not stabilize polysomes, resulting in no SG inhibition. <span style="color:blue">Blue: DAPI</span>, <span style="color:lime">green: G3BP1</span>.</p>
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<p>TLR signaling only weakly inhibits ER stress-induced SGs in iMLFs. (<b>A</b>) Effect of TLR4 stimulation using 1 µg/mL LPS for 6 h on ER stress- and sodium arsenite-induced SGs. (<b>B</b>) Quantification of SG assembly induced by ER stress. (<b>C</b>) Quantification of SG assembly induced by sodium arsenite. Abbreviations: Thap = 1 µg/mL thapsigargin for 60 min, Ars = 500 µM sodium arsenite for 60 min. <span style="color:blue">Blue: DAPI</span>, <span style="color:lime">green: G3BP1</span>, <span style="color:red">red: DDX3X</span>. * denotes <span class="html-italic">p</span> &lt; 0.05 using the two-tailed Mann–Whitney test.</p>
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<p>TLR signaling fails to inhibit SGs in primary MLFs. (<b>A</b>) Effect of TLR4 stimulation using 1 µg/mL LPS for 6 h on ER stress- and sodium arsenite-induced SGs. (<b>B</b>) Quantification of SG assembly induced by ER stress. (<b>C</b>) Quantification of SG assembly induced by sodium arsenite. Quantification of SG assembly induced by 2 µg/mL thapsigargin (Thap) treatment for 90 min in immortalized BMDMs stimulated with (<b>D</b>) LPS, (<b>E</b>) Pam3CSK4 (Pam3), (<b>F</b>) R848 and (<b>G</b>) poly(I:C) (pI:C) for 6 h. Abbreviations: Thap = 1 µg/mL thapsigargin for 60 min, Ars = 500 µM sodium arsenite for 60 min. <span style="color:blue">Blue: DAPI</span>, <span style="color:lime">green: G3BP1</span>, <span style="color:red">red: DDX3X</span>. The two-tailed Mann–Whitney test was used to calculate statistical significance. ** denotes <span class="html-italic">p</span>-value &lt; 0.01, n.s. denotes non-significant <span class="html-italic">p</span>-value (&gt;0.05).</p>
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<p>Effect of TLR4 activation on IKK kinase activity in A549 cells, MLFs, iMLFs and BMDMs. (<b>A</b>) Quantification of phosphorylated P38 (p-P38), total P38 (t-P38), phosphorylated IκBα (p-IκBα) and total IκBα (t-IκBα) in A549 cells following stimulation with 1 µg/mL LPS for indicated durations. GAPDH was used as loading control. (<b>B</b>) Quantification of phosphorylated P38 (p-P38), total P38 (t-P38), phosphorylated IκBα (p-IκBα) and total IκBα (t-IκBα) in iMLFs following stimulation with 1 µg/mL LPS for indicated durations. GAPDH was used as loading control. (<b>C</b>) Quantification of phosphorylated P38 (p-P38), total P38 (t-P38), phosphorylated IκBα (p-IκBα) and total IκBα (t-IκBα) in MLFs following stimulation with 1 µg/mL LPS for indicated durations. GAPDH was used as loading control. (<b>D</b>) Quantification of phosphorylated P38 (p-P38), total P38 (t-P38), phosphorylated IκBα (p-IκBα) and total IκBα (t-IκBα) in BMDM cells following stimulation with 100 ng/mL LPS for indicated durations. GAPDH was used as loading control. (<b>E</b>) Quantification of phosphorylated P38 (p-P38), total P38 (t-P38), phosphorylated IκBα (p-IκBα) and total IκBα (t-IκBα) in immortalized BMDM cells following stimulation with 100 ng/mL LPS for indicated durations. GAPDH was used as loading control. Abbreviations: iMLF = immortalized mouse lung fibroblast, MLF = primary mouse lung fibroblast, BMDM = bone-marrow-derived macrophage, iBMDM = immortalized bone-marrow-derived macrophage.</p>
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