Genome-wide identification of WRKY transcription factors in Casuarina equisetifolia and the function analysis of CeqWRKY11 in response to NaCl/NaHCO3 stresses.

BACKGROUND: Casuarina equisetifolia (C. equisetifolia) is a woody species with many excellent features. It has natural resistance against drought, salt and saline-alkali stresses. WRKY transcription factors (TFs) play significant roles in plant response to abiotic stresses, therefore, molecular characterization of WRKY gene family under abiotic stresses holds great significance for improvement of forest trees through molecular biological tools. At present, WRKY TFs from C. equisetifolia have not been thoroughly studied with respect to their role in salt and saline-alkali stresses response. The current study was conducted to bridge the same knowledge gap. RESULTS: A total of 64 WRKYs were identified in C. equisetifolia and divided into three major groups i.e. group I, II and III, consisting of 10, 42 and 12 WRKY members, respectively. The WRKY members in group II were further divided into 5 subgroups according to their homology with Arabidopsis counterparts. WRKYs belonging to the same group exhibited higher similarities in gene structure and the presence of conserved motifs. Promoter analysis data showed the presence of various response elements, especially those related to hormone signaling and abiotic stresses, such as ABRE (ABA), TGACG (MeJA), W-box ((C/T) TGAC (T/C)) and TC-rich motif. Tissue specific expression data showed that CeqWRKYs were mainly expressed in root under normal growth conditions. Furthermore, most of the CeqWRKYs were up-regulated by NaCl and NaHCO3 stresses with few of WRKYs showing early responsiveness to both stresses while few others exhibiting late response. Although the expressions of CeqWRKYs were also induced by cold stress, the response was delayed compared with other stresses. Transgenic C. equisetifolia plants overexpressing CeqWRKY11 displayed lower electrolyte leakage, higher chlorophyll content, and enhanced tolerance to both stresses. The higher expression of abiotic stress related genes, especially CeqHKT1 and CeqPOD7, in overexpression lines points to the maintenance of optimum Na+/K+ ratio, and ROS scavenging as possible key molecular mechanisms underlying salt stress tolerance. CONCLUSIONS: Our results show that CeqWRKYs might be key regulators of NaCl and NaHCO3 stresses response in C. equisetifolia. In addition, positive correlation of CeqWRKY11 expression with increased stress tolerance in C. equisetifolia encourages further research on other WRKY family members through functional genomic tools. The best candidates could be incorporated in other woody plant species for improving stress tolerance.

Preliminary investigation into long-term stress by isolated captivity-related changes of reproduction hormones in Cynomolgus monkey.

BACKGROUND: Stress profoundly affects physical and emotional well-being, extending its physiological influence to the female menstrual cycle, impeding the hypothalamus-pituitary-gonadal (HPG) axis, and affecting fertility by suppressing sex-stimulating hormones. METHODS: In this study, we meticulously analyzed menstrual cycles and corresponding hormonal fluctuations in three female Cynomolgus monkeys. RESULTS: The preliminary findings indicated lower-than-normal levels of cortisol, follicle-stimulating hormone (FSH), and estradiol. Anovulatory bleeding occurred in one monkey, which could be linked to stress. In contrast to cortisol, alkaline phosphatase (ALP), which is correlated to cortisol levels, was consistently elevated in menstruating monkeys, suggesting its potential as a stress indicator. The non-menstruating group exhibited stress-related weight loss, emphasizing the observed ALP trends. CONCLUSIONS: Non-menstruating monkeys may experience more stress than menstruating monkeys. The implications of this study extend beyond the confines of primate studies and offer a valuable method for enhancing the welfare of female Cynomolgus monkeys.

Genome-wide identification of the plant homeodomain-finger family in rye and ScPHD5 functions in cold tolerance and flowering time.

KEY MESSAGE: 111 PHD genes were newly identified in rye genome and ScPHD5's role in regulating cold tolerance and flowering time was suggested. Plant homeodomain (PHD)-finger proteins regulate the physical properties of chromatin and control plant development and stress tolerance. Although rye (Secale cereale L.) is a major winter crop, PHD-finger proteins in rye have not been studied. Here, we identified 111 PHD genes in the rye genome that exhibited diverse gene and protein sequence structures. Phylogenetic tree analysis revealed that PHDs were genetically close in monocots and diverged from those in dicots. Duplication and synteny analyses demonstrated that ScPHDs have undergone several duplications during evolution and that high synteny is conserved among the Triticeae species. Tissue-specific and abiotic stress-responsive gene expression analyses indicated that ScPHDs were highly expressed in spikelets and developing seeds and were responsive to cold and drought stress. One of these genes, ScPHD5, was selected for further functional characterization. ScPHD5 was highly expressed in the spike tissues and was localized in the nuclei of rye protoplasts and tobacco leaves. ScPHD5-overexpressing Brachypodium was more tolerant to freezing stress than wild-type (WT), with increased CBF and COR gene expression. Additionally, these transgenic plants displayed an extremely early flowering phenotype that flowered more than two weeks earlier than the WT, and vernalization genes, rather than photoperiod genes, were increased in the WT. RNA-seq analysis revealed that diverse stress response genes, including HSPs, HSFs, LEAs, and MADS-box genes, were also upregulated in transgenic plants. Our study will help elucidate the roles of PHD genes in plant development and abiotic stress tolerance in rye.

Overcoming Nutrient Stress: Integrin αvß3-Driven Metabolic Adaptation Supports Tumor Initiation.

Nutrient stress accompanies several stages of tumor progression, including metastasis formation. Metabolic reprogramming is a hallmark of cancer, and it has been associated with stress tolerance and anchorage-independent cell survival. Adaptive responses are required to support cancer cell survival under these conditions. In this issue of Cancer Research, Nam and colleagues showed that the extracellular matrix (ECM) receptor integrin ß3 was upregulated in lung cancer cells in response to nutrient starvation, resulting in increased cell survival that was independent from ECM binding. Delving into the molecular mechanisms responsible for this, the authors found that integrin ß3 promoted glutamine metabolism and oxidative phosphorylation (OXPHOS) by activating a Src/AMPK/PGC1α signaling pathway. Importantly, in vivo experiments confirmed that OXPHOS inhibition suppressed tumor initiation in an orthotopic model of lung cancer, while ß3 knockout completely abrogated tumor initiation. These observations indicate that targeting signaling pathways downstream of αvß3 could represent a promising therapeutic avenue to prevent lung cancer progression and metastasis. See related article by Nam et al., p. 1630.

Selection of rice breeding lines for resistance to biotic and abiotic stresses.

Rice (Oryza sativa L.) grown in many countries around the world with different climatic conditions and a huge number of environmental stresses, both biotic (fungi, bacteria, viruses, insects) and abiotic (cold, drought, salinity) limit rice productivity. In this regard, breeders and scientists are trying to create rice lines that are resistant to multiple stresses. The aim of this work was to screen and select cold and blast resistant rice breeding lines (RBLs) using molecular markers. Molecular screening of RBLs and parental varieties to cold tolerance was carried out using markers RM24545, RM1377, RM231 and RM569 associated with QTLs (qPSST-3, qPSST-7, qPSST-9). It was discovered that the presence of three QTLs characterizes the cold resistance of studied genotypes, and the absence of one of them leads to cold sensitivity. As a result, 21 cold-resistant out of the 28 studied RBLs were identified. These cold resistant 21 RBLs were further tested to blast resistance using markers Pi-ta, Pita3, Z56592, 195R-1, NMSMPi9-1, TRS26, Pikh MAS, MSM6, 9871.T7E2b, RM224 and RM1233. It was revealed that 16 RBLs from 21 studied lines contain 5-6 blast resistance genes. In accordance with the blast resistance strategy, the presence of 5 or more genes ensures the formation of stable resistance to Magnaporthe oryzae. Thus, 16 lines resistant to multiple stresses, such as cold and blast disease were developed. It should be noted that 6 of these selected lines are high-yielding, which is very important in rice breeding program. These RBLs can be used in breeding process as starting lines, germplasm exchange as a source of resistant genes for the development of new rice varieties resistant to multiple stress factors.

Transcriptome dynamics of Gossypium purpurascens in response to abiotic stresses by Iso-seq and RNA-seq data.

Gossypium purpurascens is a member of the Malvaceae family, holds immense economic significance as a fiber crop worldwide. Abiotic stresses harm cotton crops, reduce yields, and cause economic losses. Generating high-quality reference genomes and large-scale transcriptomic datasets across diverse conditions can offer valuable insights into identifying preferred agronomic traits for crop breeding. The present research used leaf tissues to conduct PacBio Iso-seq and RNA-seq analysis. We carried out an in-depth analysis of DEGs using both correlations with cluster analysis and principal component analysis. Additionally, the study also involved the identification of both lncRNAs and CDS. We have prepared RNA-seq libraries from 75 RNA samples to study the effects of drought, salinity, alkali, and saline-alkali stress, as well as control conditions. A total of 454.06 Gigabytes of transcriptome data were effectively validated through the identification of differentially expressed genes and KEGG and GO analysis. Overwhelmingly, gene expression profiles and full-length transcripts from cotton tissues will aid in understanding the genetic mechanism of abiotic stress tolerance in G. purpurascens.

Intracellular Protective Functions and Therapeutical Potential of Trehalose.

Trehalose is a naturally occurring, non-reducing saccharide widely distributed in nature. Over the years, research on trehalose has revealed that this initially thought simple storage molecule is a multifunctional and multitasking compound protecting cells against various stress factors. This review presents data on the role of trehalose in maintaining cellular homeostasis under stress conditions and in the virulence of bacteria and fungi. Numerous studies have demonstrated that trehalose acts in the cell as an osmoprotectant, chemical chaperone, free radical scavenger, carbon source, virulence factor, and metabolic regulator. The increasingly researched medical and therapeutic applications of trehalose are also discussed.

Cadmium binding by the F-box domain induces p97-mediated SCF complex disassembly to activate stress response programs.

The F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCFMet30 ubiquitin ligase during cadmium stress. Several highly conserved cysteine residues within the Met30 F-box contribute to binding of cadmium with a KD of 8 µM. Binding induces a conformational change that allows for Met30 autoubiquitylation, which in turn leads to recruitment of the segregase Cdc48/p97/VCP followed by active SCFMet30 disassembly. The resulting inactivation of SCFMet30 protects cells from cadmium stress. Our results show that F-box domains participate in regulation of SCF ligases beyond formation of the Skp1 binding interface.

Elucidating the role of exogenous melatonin in mitigating alkaline stress in soybeans across different growth stages: a transcriptomic and metabolomic approach.

BACKGROUND: Soybean (Glycine max), a vital grain and oilseed crop, serves as a primary source of plant protein and oil. Soil salinization poses a significant threat to soybean planting, highlighting the urgency to improve soybean resilience and adaptability to saline stress. Melatonin, recently identified as a key plant growth regulator, plays crucial roles in plant growth, development, and responses to environmental stress. However, the potential of melatonin to mitigate alkali stress in soybeans and the underlying mechanisms remain unclear. RESULTS: This study investigated the effects of exogenous melatonin on the soybean cultivar Zhonghuang 13 under alkaline stress. We employed physiological, biochemical, transcriptomic, and metabolomic analyses throughout both vegetative and pod-filling growth stages. Our findings demonstrate that melatonin significantly counteracts the detrimental effects of alkaline stress on soybean plants, promoting plant growth, photosynthesis, and antioxidant capacity. Transcriptomic analysis during both growth stages under alkaline stress, with and without melatonin treatment, identified 2,834 and 549 differentially expressed genes, respectively. These genes may play a vital role in regulating plant adaptation to abiotic stress. Notably, analysis of phytohormone biosynthesis pathways revealed altered expression of key genes, particularly in the ARF (auxin response factor), AUX/IAA (auxin/indole-3-acetic acid), and GH3 (Gretchen Hagen 3) families, during the early stress response. Metabolomic analysis during the pod-filling stage identified highly expressed metabolites responding to melatonin application, such as uteolin-7-O-(2''-O-rhamnosyl)rutinoside and Hederagenin-3-O-glucuronide-28-O-glucosyl(1,2)glucoside, which helped alleviate the damage caused by alkali stress. Furthermore, we identified 183 differentially expressed transcription factors, potentially playing a critical role in regulating plant adaptation to abiotic stress. Among these, the gene SoyZH13_04G073701 is particularly noteworthy as it regulates the key differentially expressed metabolite, the terpene metabolite Hederagenin-3-O-glucuronide-28-O-glucosyl(1,2)glucoside. WGCNA analysis identified this gene (SoyZH13_04G073701) as a hub gene, positively regulating the crucial differentially expressed metabolite of terpenoids, Hederagenin-3-O-glucuronide-28-O-glucosyl(1,2)glucoside. Our findings provide novel insights into how exogenous melatonin alleviates alkali stress in soybeans at different reproductive stages. CONCLUSIONS: Integrating transcriptomic and metabolomic approaches, our study elucidates the mechanisms by which exogenous melatonin ameliorates the inhibitory effects of alkaline stress on soybean growth and development. This occurs through modulation of biosynthesis pathways for key compounds, including terpenes, flavonoids, and phenolics. Our findings provide initial mechanistic insights into how melatonin mitigates alkaline stress in soybeans, offering a foundation for molecular breeding strategies to enhance salt-alkali tolerance in this crop.

Mycobacterium tuberculosis PhoP integrates stress response to intracellular survival by regulating cAMP level.

Survival of Mycobacterium tuberculosis within the host macrophages requires the bacterial virulence regulator PhoP, but the underlying reason remains unknown. 3',5'-Cyclic adenosine monophosphate (cAMP) is one of the most widely used second messengers, which impacts a wide range of cellular responses in microbial pathogens including M. tuberculosis. Herein, we hypothesized that intra-bacterial cAMP level could be controlled by PhoP since this major regulator plays a key role in bacterial responses against numerous stress conditions. A transcriptomic analysis reveals that PhoP functions as a repressor of cAMP-specific phosphodiesterase (PDE) Rv0805, which hydrolyzes cAMP. In keeping with these results, we find specific recruitment of the regulator within the promoter region of rv0805 PDE, and absence of phoP or ectopic expression of rv0805 independently accounts for elevated PDE synthesis, leading to the depletion of intra-bacterial cAMP level. Thus, genetic manipulation to inactivate PhoP-rv0805-cAMP pathway decreases cAMP level, stress tolerance, and intracellular survival of the bacillus.

α,ß-trehalose, an intracellular substance in resting cyst of colpodid ciliates as a key to environmental tolerances.

α,α-trehalose is a well-known sugar that plays a key role in establishing tolerance to environmental stresses in many organisms, except unicellular eukaryotes. However, almost nothing is known about α,ß-trehalose, including their synthesis, function, and even presence in living organisms. In this study, we identified α,ß-trehalose in the resting cyst, a dormancy cell form characterized by extreme tolerance to environmental stresses, of the ciliated protist Colpoda cucullus, using high-performance liquid chromatography (HPLC), and a proton nuclear magnetic resonance (1H NMR). Gene expression analysis revealed that the expression of trehalose-6-phosphate synthase (TPS), glycosyltransferase (GT), alpha-amylase (AMY), and trehalose transporter 1 (TRET1), were up-regulated in encystment, while the expression of α-glucosidase 2 (AG2) and trehalase (TREH) was up-regulated in excystment. These results suggest that α,ß-trehalose is synthesized during encystment process, while and contributes to extreme tolerances to environmental stressors, stored carbohydrates, and energy reserve during resting cyst and/or during excystment.

Integrated transcriptomics and metabolomics revealed the mechanism of catechin biosynthesis in response to lead stress in tung tree (Vernicia fordii).

Lead (Pb) affects gene transcription, metabolite biosynthesis and growth in plants. The tung tree (Vernicia fordii) is highly adaptive to adversity, whereas the mechanisms underlying its response to Pb remain uncertain. In this work, transcriptomic and metabolomic analyses were employed to study tung trees under Pb stress. The results showed that the biomass of tung seedlings decreased with increasing Pb doses, and excessive Pb doses resulted in leaf wilting, root rot, and disruption of Pb homeostasis. Under non-excessive Pb stress, a significant change in the expression patterns of flavonoid biosynthesis genes was observed in the roots of tung seedlings, leading to changes in the accumulation of flavonoids in the roots, especially the upregulation of catechins, which can chelate Pb and reduce its toxicity in plants. In addition, Pb-stressed roots showed a large accumulation of VfWRKY55, VfWRKY75, and VfLRR1 transcripts, which were shown to be involved in the flavonoid biosynthesis pathway by gene module analysis. Overexpression of VfWRKY55, VfWRKY75, and VfLRR1 significantly increased catechin concentrations in tung roots, respectively. These data indicate that Pb stress-induced changes in the expression patterns of those genes regulate the accumulation of catechins. Our findings will help to clarify the molecular mechanism of Pb response in plants.

Cold pressor stress effects on cardiac repolarization.

The cold pressor test (CPT) elicits strong cardiovascular reactions via activation of the sympathetic nervous system (SNS), yielding subsequent increases in heart rate (HR) and blood pressure (BP). However, little is known on how exposure to the CPT affects cardiac ventricular repolarization. Twenty-eight healthy males underwent both a bilateral feet CPT and a warm water (WW) control condition on two separate days, one week apart. During pre-stress baseline and stress induction cardiovascular signals (ECG lead II, Finometer BP) were monitored continuously. Salivary cortisol and subjective stress ratings were assessed intermittently. Corrected QT (QTc) interval length and T-wave amplitude (TWA) were assessed for each heartbeat and subsequently aggregated individually over baseline and stress phases, respectively. CPT increases QTc interval length and elevates the TWA. Stress-induced changes in cardiac repolarization are only in part and weakly correlated with cardiovascular and cortisol stress-reactivity. Besides its already well-established effects on cardiovascular, endocrine, and subjective responses, CPT also impacts on cardiac repolarization by elongation of QTc interval length and elevation of TWA. CPT effects on cardiac repolarization share little variance with the other indices of stress reactivity, suggesting a potentially incremental value of this parameter for understanding psychobiological adaptation to acute CPT stress.

Carbon substrates promotes stress resistance and drug tolerance in clinical isolates of Candida tropicalis.

Candida tropicalis is a human pathogen and one of the most prevalent non-Candida albicans Candida (NCAC) species causing invasive infections. Azole antifungal resistance in C. tropicalis is also gradually increasing with the increasing incidence of infections. The pathogenic success of C. tropicalis depends on its effective response in the host microenvironment. To become a successful pathogen, cellular metabolism, and physiological status determine the ability of the pathogen to counter diverse stresses inside the host. However, to date, limited knowledge is available on the impact of carbon substrate metabolism on stress adaptation and azole resistance in C. tropicalis. In this study, we determined the impact of glucose, fructose, and sucrose as the sole carbon source on the fluconazole resistance and osmotic (NaCl), oxidative (H2O2) stress adaptation in C. tropicalis clinical isolates. We confirmed that the abundance of carbon substrates influences or increases drug resistance and osmotic and oxidative stress tolerance in C. tropicalis. Additionally, both azole-resistant and susceptible isolates showed similar stress adaptation phenotypes, confirming the equal efficiency of becoming successful pathogens irrespective of drug susceptibility profile. To the best of our knowledge, our study is the first on C. tropicalis to demonstrate the direct relation between carbon substrate metabolism and stress tolerance or drug resistance.

Enhancing iron content and growth of cucumber seedlings with MgFe-LDHs under low-temperature stress.

The development of cost-effective and eco-friendly fertilizers is crucial for enhancing iron (Fe) uptake in crops and can help alleviate dietary Fe deficiencies, especially in populations with limited access to meat. This study focused on the application of MgFe-layered double hydroxide nanoparticles (MgFe-LDHs) as a potential solution. We successfully synthesized and characterized MgFe-LDHs and observed that 1-10 mg/L MgFe-LDHs improved cucumber seed germination and water uptake. Notably, the application of 10 mg/L MgFe-LDHs to roots significantly increased the seedling emergence rate and growth under low-temperature stress. The application of 10 mg/L MgFe-LDHs during sowing increased the root length, lateral root number, root fresh weight, aboveground fresh weight, and hypocotyl length under low-temperature stress. A comprehensive analysis integrating plant physiology, nutrition, and transcriptomics suggested that MgFe-LDHs improve cold tolerance by upregulating SA to stimulate CsFAD3 expression, elevating GA3 levels for enhanced nitrogen metabolism and protein synthesis, and reducing levels of ABA and JA to support seedling emergence rate and growth, along with increasing the expression and activity of peroxidase genes. SEM and FTIR further confirmed the adsorption of MgFe-LDHs onto the root hairs in the mature zone of the root apex. Remarkably, MgFe-LDHs application led to a 46% increase (p < 0.05) in the Fe content within cucumber seedlings, a phenomenon not observed with comparable iron salt solutions, suggesting that the nanocrystalline nature of MgFe-LDHs enhances their absorption efficiency in plants. Additionally, MgFe-LDHs significantly increased the nitrogen (N) content of the seedlings by 12% (p < 0.05), promoting nitrogen fixation in the cucumber seedlings. These results pave the way for the development and use of LDH-based Fe fertilizers.

A meta-analysis of photosynthetic efficiency and stress mitigation by melatonin in enhancing wheat tolerance.

BACKGROUND: Our meta-analysis examines the effects of melatonin on wheat under varying abiotic stress conditions, focusing on photosynthetic parameters, chlorophyll fluorescence, leaf water status, and photosynthetic pigments. We initially collected 177 publications addressing the impact of melatonin on wheat. After meticulous screening, 31 published studies were selected, encompassing 170 observations on photosynthetic parameters, 73 on chlorophyll fluorescence, 65 on leaf water status, 240 on photosynthetic pigments. RESULTS: The analysis revealed significant heterogeneity across studies (I² > 99.90%) for the aforementioned parameters and evidence of publication bias, emphasizing the complex interaction between melatonin application and plant physiological responses. Melatonin enhanced the overall response ratio (lnRR) for photosynthetic rates, stomatal conductance, transpiration rates, and fluorescence yields by 20.49, 22.39, 30.96, and 1.09%, respectively, compared to the control (no melatonin). The most notable effects were under controlled environmental conditions. Moreover, melatonin significantly improved leaf water content and reduced water potential, particularly under hydroponic conditions and varied abiotic stresses, highlighting its role in mitigating water stress. The analysis also revealed increases in chlorophyll pigments with soil drenching and foliar spray, and these were considered the effective application methods. Furthermore, melatonin influenced chlorophyll SPAD and intercellular CO2 concentrations, suggesting its capacity to optimize photosynthetic efficiency. CONCLUSIONS: This synthesis of meta-analysis confirms that melatonin significantly enhances wheat's resilience to abiotic stress by improving photosynthetic parameters, chlorophyll fluorescence, leaf water status, and photosynthetic pigments. Despite observed heterogeneity and publication bias, the consistent beneficial effects of melatonin, particularly under controlled conditions with specific application methods e.g. soil drenching and foliar spray, demonstrate its utility as a plant growth regulator for stress management. These findings encourage focused research and application strategies to maximize the benefits of melatonin in wheat farming, and thus contributing to sustainable agricultural practices.

Systems theory: A novel approach for understanding how stressors affect honey bee health 'all at once'.

A new study showcases the usefulness of systems theory and network analyses for understanding how dozens of stressors can act concomitantly to affect managed honey bee health. Interestingly, the most influential stressors are not those currently being addressed by beekeepers.

Feather corticosterone is lower in translocated and historical populations of the endangered Laysan duck (Anas laysanensis).

Identifying reliable bioindicators of population status is a central goal of conservation physiology. Physiological stress measures are often used as metrics of individual health and can assist in managing endangered species if linked to fitness traits. We analysed feather corticosterone, a cumulative physiological stress metric, of individuals from historical, translocated, and source populations of an endangered endemic Hawaiian bird, the Laysan duck (Anas laysanensis). We hypothesized that feather corticosterone would reflect the improved reproduction and survival rates observed in populations translocated to Midway and Kure Atolls from Laysan Island. We also predicted less physiological stress in historical Laysan birds collected before ecological conditions deteriorated and the population bottleneck. All hypotheses were supported: we found lower feather corticosterone in the translocated populations and historical samples than in those from recent Laysan samples. This suggests that current Laysan birds are experiencing greater physiological stress than historical Laysan and recently translocated birds. Our initial analysis suggests that feather corticosterone may be an indicator of population status and could be used as a non-invasive physiological monitoring tool for this species with further validation. Furthermore, these preliminary results, combined with published demographic data, suggest that current Laysan conditions may not be optimal for this species.

Analysis of Cellular Stress Assay Parameters and Intracellular ATP in Platelets: Comparison of Platelet Preparation Methods.

Platelets are metabolically active, anucleated and small circulating cells mainly responsible for the prevention of bleeding and maintenance of hemostasis. Previous studies showed that platelets mitochondrial content, function, and energy supply change during several diseases such as HIV/AIDS, COVID-19, pulmonary arterial hypertension, and in preeclampsia during pregnancy. These changes in platelets contributed to the severity of diseases and mortality. In our previous studies, we have shown that the seahorse-based cellular stress assay (CSA) parameters are crucial to the understanding of the mitochondrial performance in peripheral blood mononuclear cells (PBMCS). Moreover, the results of CSA parameters were significantly influenced by the PBMC preparation methods. In this study, we assessed the correlation of CSA parameters and intracellular ATP content in platelets and evaluated the effects of platelet preparation methods on the results of CSA parameters and intracellular ATP content. We compared the results of CSA parameters and intracellular ATP content in platelets isolated by density centrifugation with Optiprep and simple centrifugation of blood samples without Optiprep. Platelets isolated by centrifugation with Optiprep showed a higher spare capacity, basal respiration, and maximal respiration than those isolated without Optiprep. There was a clear correlation between basal respiration and maximal respiration, and the whole-ATP content in both isolation methods. Moreover, a positive correlation was observed between the relative spare capacity and whole-cell ATP content. In conclusion, the results of seahorse-based CSA parameters and intracellular ATP content in platelets are markedly influenced by the platelet isolation methods employed. The results of basal respiration and maximal respiration are hallmarks of cellular activity in platelets, and whole-cell ATP content is a potential hint for basic platelet viability. We recommend further studies to evaluate the role of CSA parameters and intracellular ATP content in platelets as biomarkers for the diagnosis and prediction of disease states.

Stress-Induced Proteasome Sub-Cellular Translocation in Cardiomyocytes Causes Altered Intracellular Calcium Handling and Arrhythmias.

The ubiquitin-proteasome system (UPS) is an essential mechanism responsible for the selective degradation of substrate proteins via their conjugation with ubiquitin. Since cardiomyocytes have very limited self-renewal capacity, as they are prone to protein damage due to constant mechanical and metabolic stress, the UPS has a key role in cardiac physiology and pathophysiology. While altered proteasomal activity contributes to a variety of cardiac pathologies, such as heart failure and ischemia/reperfusion injury (IRI), the environmental cues affecting its activity are still unknown, and they are the focus of this work. Following a recent study by Ciechanover's group showing that amino acid (AA) starvation in cultured cancer cell lines modulates proteasome intracellular localization and activity, we tested two hypotheses in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs, CMs): (i) AA starvation causes proteasome translocation in CMs, similarly to the observation in cultured cancer cell lines; (ii) manipulation of subcellular proteasomal compartmentalization is associated with electrophysiological abnormalities in the form of arrhythmias, mediated via altered intracellular Ca2+ handling. The major findings are: (i) starving CMs to AAs results in proteasome translocation from the nucleus to the cytoplasm, while supplementation with the aromatic amino acids tyrosine (Y), tryptophan (W) and phenylalanine (F) (YWF) inhibits the proteasome recruitment; (ii) AA-deficient treatments cause arrhythmias; (iii) the arrhythmias observed upon nuclear proteasome sequestration(-AA+YWF) are blocked by KB-R7943, an inhibitor of the reverse mode of the sodium-calcium exchanger NCX; (iv) the retrograde perfusion of isolated rat hearts with AA starvation media is associated with arrhythmias. Collectively, our novel findings describe a newly identified mechanism linking the UPS to arrhythmia generation in CMs and whole hearts.