Lower ROS Levels Research Articles

Predictive Indicators of Cryotolerance and Fertility in Bovine Sperm: Evaluating Fresh Semen Quality to Improve AI Outcomes With Frozen-Thawed Sperm.

The success of artificial insemination (AI) with frozen-thawed semen in cattle is influenced by both female factors and sperm quality. In terms of sperm quality, prior studies indicate that the ability of frozen-thawed bovine sperm to fertilise an oocyte is dependent on their quality and resilience to cryopreservation. Cryopreservation induces oxidative stress, leading to ultrastructural damage in the sperm. This study aimed to determine whether the quality of fresh semen can identify bulls with good and poor sperm freezability. This difference between fresh and frozen semen from the same bull allows us to predict fertility. Motility and kinetic parameters were assessed using computer-assisted sperm analysis (CASA), while six functional variables were evaluated through flow cytometry, both before and after the freeze-thaw process on the sperm from 13 bulls. Invivo fertility was measured using 90-day non-return rates. The principal component analysis (PCA) of eight sperm variables post-thaw identified one principal component explaining 81.19% of the total variance and classified the bulls into two groups: Poor freezability bulls (progressive motility: 48.12% ± 8.41%; viability: 77.51% ± 7.61%) and good freezability bulls (progressive motility: 58.64% ± 6.64%; viability: 88.12% ± 2.52%). Bulls with higher freezability showed better sperm viability and motility, as well as lower levels of ROS, superoxides and intracellular calcium before cryopreservation that were significantly correlated with higher non-return rates (NRR). The results underscore the importance of assessing the quality and functionality of fresh semen to predict the fertility potential of cryopreserved sperm. This approach can aid in selecting ejaculates with the best potential for successful artificial insemination, ultimately improving reproductive performance in dairy cattle.

Regulation of the RCK1 gene on the oxidative tolerance of Saccharomyces cerevisiae

Our previous work indicated that the quorum sensing (QS) effect could regulate the oxidative tolerance of Saccharomyces cerevisiae, and QS may impact oxidative and antioxidative metabolisms of S. cerevisiae by regulating the RCK1 gene. Therefore, this work proposed a reasonable logic that RCK1 could play roles in regulating the oxidative and antioxidative metabolisms of yeast cells. The results presented here suggested that the overexpression of RCK1 has a regulatory effect on the reduction of ROS level and the promotion of oxidative tolerance of S. cerevisiae. The overexpression of RCK1 promoted the ROS generation through activating the MAPK pathway; on the other hand, RCK1-regulated antioxidative metabolism played a more significant role to realize lower ROS level and higher oxidative tolerance of S288c-RCK1 and ΔARO80-RCK1 strains. To improve the fermentation performance of yeast while circumventing metabolic burden, a recombinant strain with over time-controlled overexpression of the RCK1 gene (i.e., S288c'-RCK1 strain) derived from S288c strain was successfully constructed to achieve artificial regulation of yeast oxidative tolerance. Transcriptomics analysis was further performed on both S. cerevisiae wild-type and S288c'-RCK1 strains to identify differentially expressed genes and analyze their functional pathway classification. This work is instructive for artificially modulating the oxidative tolerance of strains to enhance the fermentation performance of yeast.

Angiotensin-converting enzyme 2 activation attenuates inflammation and oxidative stress in brain death donor followed by rat lung transplantation

Brain death (BD) provides most of the donor organs destined for lung transplantation (LTx). However, the organs may be affected by inflammatory and oxidative processes. Based on this, we hypothesize that the angiotensin-converting enzyme 2 (ACE2) activation can reduce the lung injury associated with LTx. 3 h after BD induction, rats were injected with saline (BD group) or an ACE2 activator (ACE2a group; 15 mg/kg-1) and kept on mechanical ventilation for additional 3 h. A third group included a control ventilation (Control group) prior to transplant. After BD protocol, left LTx were performed, followed by 2 h-reperfusion. ACE2 activation was associated with better oxygenation after BD management (p = 0.01), attenuating edema (p = 0.05) followed by the reduction in tissue resistance (p = 0.01) and increase of respiratory compliance (p = 0.02). Nrf2 expression was also upregulated in the ACE2a group (p = 0.03). After transplantation, ACE2a group showed lower levels of TNF-α (p = 0.02), IL-6 (p = 0.001), IL-1β (p = 0.01), ROS (p = 0.004) and MDA (p = 0.002), in addition to higher CAT activity (p = 0.04). In conclusion, our study suggests that ACE2 activation improves anti-inflammatory and antioxidant activity in a model of LTx.

Dietary supplement guava leaf extract regulates growth, feed utilization, immune function, nutrient digestibility and redox regulation in growing rabbits

The use of agricultural waste in animal production has gained global interest. An eight-week trial was conducted to investigate the impacts of adding ethanolic guava leaf extract (GLE) as a feed supplement on the growth, feed utilization, immune response, nutrient digestibility, redox regulation, and blood health of growing rabbits. Ninety weaned growing rabbits were randomly assigned to three groups. The first group was fed a basal diet (GLE0), while the other two groups were fed the control diet fortified with 15 mg (GLE15) or 20 mg (GLE20) of GLE per kg of diet for 8 weeks. The HPLC analysis of GLE exhibited the presence of gallic acid, ferulic acid, catechin, and caffeic acid in significant amounts. The results indicated that final body weight, daily body weight, daily feed intake and nutrient digestibility were significantly higher in the GLE-treated groups compared to the un-treated group (p < 0.05). Dietary supplementation of GLE significantly reduced lipid contents including triglycerides, total cholesterol, LDL, HDL, and VLDL (P < 0.05), with the most significant results observed when adding 20 mg/kg to the diet. AST and ALT levels as well as cortisol hormone in rabbits fed GLE were lower than those in the GLE0 group (P < 0.05). Immunoglobulins (IgG and IgA), antioxidant biomarkers (SOD and TAC) and T3 hormone were significantly improved by GLE supplementation (P < 0.001). Rabbits fed with GLE had lower levels of ROS and MDA compared to those in the GLE0 group (P < 0.001). Moreover, the hepatic and intestinal architectures were maintained in all rabbits fed diets with GLE. The results suggest that GLE supplementation (20 mg/kg diet) in fattening rabbit diets could efficiently improve growth, health status, blood physiology, antioxidant capacity and tissue histology.

Hypoxia-inducible factor-1α promotes ferroptosis by inducing ferritinophagy and promoting lactate production in yak longissimus thoracis et lumborum postmortem

Ferroptosis has emerged as a novel, crucial regulator of meat quality in the postmortem hypoxia environment, with its role in mediating protein oxidation and cell death. However, the interaction between ferroptosis and the hypoxia response, especially the involvement of hypoxia-inducible factor-1α (HIF-1α), remains poorly studied. This study aimed to characterize whether HIF-1α influences ferroptosis, and, if so, explore the underlying mechanisms involved. The results showed that ferroptosis mediated by HIF-1α negatively impacts meat color and water holding capacity (WHC) but improving tenderness. Inhibition of HIF-1α by 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) reduced ferroptosis, as evidenced by lower lipid ROS levels, malondialdehyde (MDA), along with higher glutathione (GSH) levels compared to the control (P < 0.05). Additionally, inhibition of HIF-1α shifted iron homeostasis towards decreased uptake via downregulation of transferrin receptor 1 (TfR1) and induced export/storage via upregulation of ferroportin (FPN) and ferritin heavy chain (FTH) (P < 0.05). The relative expression of the ferritinophagy mediator nuclear receptor coactivator 4 (NCOA4), LC3-II/LC3-I ratio, and ATG were inhibited by YC-1 (P < 0.05), these findings suggest a general decrease in ferritinophagy associated with HIF-1α inhibition. YC-1-treated samples exhibited significantly diminished lactate accumulation and lactate dehydrogenase (LDH) activity compared to the control (P < 0.05). Unexpectedly, the inhibition of ferroptosis caused by YC-1 was further amplified by lactate enhancement, suggesting that lactate can exert its suppressive effects on ferroptosis independently of HIF-1α. Collectively, these findings demonstrate that HIF-1α drives ferroptosis by regulating iron metabolism, while lactate inhibits ferroptosis in a HIF-1α-independent manner. Overall, the HIF-1α mediated ferroptosis of postmortem yak muscle had a negative impact on WHC and color, while as a contributing factor of tenderness.

Response of seed germination, seedling growth and physiological characteristics to alkali stress in halophyte Suaeda liaotungensis.

Soil salinization has been considered as a major environmental threat to plant growth. Different types of salt in saline soil have different effects on germination and seedling growth. Effect of NaCl on germination and seedling establishment in Suaeda liaotungensis have been reported, but its response to alkali stress remains unclear. Our results showed that brown seeds had higher germination rate, however, black seeds had higher germination recovery percentage under alkali stress. Na2CO3 had stronger inhibitory effect on germination and seedling growth than NaHCO3. As the concentration of alkali stress increased, the ROS level of brown seeds gradually ascended, while that of black seeds decreased first and then ascended. MDA content of dimorphic seeds significantly increased under alkali stress. The trend of SOD, POD and CAT activity between dimorphic seeds was similar under the same type of alkali stress. Alkali stress enhanced proline content of dimorphic seeds, and dimorphic seeds in NaHCO3 solution had higher proline content than Na2CO3 solution. Moreover, radicle and shoot tolerance indexes of seedlings in NaHCO3 solution were significantly higher than that of Na2CO3 solution. Under strong alkali stress, seedlings in NaHCO3 solution had significantly lower ROS level and MDA content as well as higher antioxidant enzyme activity than Na2CO3 solution. This study comprehensively compared the morphological and physiological characteristics in germination and seedlings to better reveal the saline-alkali tolerance mechanisms in S. liaotungensis.

Managing Oxidative Stress Using Vitamin C to Improve Biocompatibility of Polycaprolactone for Bone Regeneration In Vitro.

Increased oxidative stress in bone cells is known to negatively alter favorable bone regeneration. This study aimed to develop a porous polycaprolactone (PCL) membrane incorporated with 25 wt % Vitamin C (PCL-Vit C) and compared it to the PCL membrane to control oxidative stress and enhance biomineralization in vitro. Both membranes were characterized using SEM-EDS, FTIR spectroscopy, and surface hydrophilicity. Vitamin C release was quantified colorimetrically. Assessments of the viability and attachment of human fetal osteoblast (hFOB 1.19) cells were carried out using XTT assay, SEM, and confocal microscopy, respectively. ROS generation and wound healing percentage were measured using flow cytometry and ImageJ software, respectively. Mineralization study using Alizarin Red in the presence or absence of osteogenic media was carried out to measure the calcium content. Alkaline phosphatase assay and gene expression of osteogenic markers (alkaline phosphatase (ALP), collagen Type I (Col1), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN)) were analyzed by real-time PCR. SEM images revealed smooth, fine, bead-free fibers in both membranes. The FTIR spectrum of pure vitamin C was replaced with peaks at 3436.05 and 2322.83 cm-1 in the PCL-Vit C membrane. Vitamin C release was detected at 15 min and 1 h. The PCL-Vit C membrane was hydrophilic, generated lower ROS, and showed significantly higher viability than the PCL membrane. Although both PCL and PCL-Vit C membranes showed similar cellular and cytoskeletal morphology, more cell clusters were evident in the PCL-Vit C membrane. Lower ROS level in the PCL-Vit C membrane displayed improved cell functionality as evidenced by enhanced cellular differentiation with more intense alizarin staining and higher calcium content, supported by upregulation of osteogenic markers ALP, Col1, and OPN even in the absence of osteogenic supplements. The presence of Vitamin C in the PCL-Vit C membrane may have mitigated oxidative stress in hFOB 1.19 cells, resulting in enhanced biomineralization facilitating bone regeneration.

Caloric restriction leading to attenuation of experimental Alzheimer's disease results from alterations in gut microbiome.

Caloric restriction (CR) might be effective for alleviating/preventing Alzheimer's disease (AD), but the biological mechanisms remain unclear. In the current study, we explored whether CR caused an alteration of gut microbiome and resulted in the attenuation of cognitive impairment of AD animal model. Thirty-week-old male APP/PS1 transgenic mice were used as AD models (AD mouse). CR was achieved by 30% reduction of daily free feeding (ad libitum, AL) amount. The mice were fed with CR protocol or AL protocol for six consecutive weeks. We found that with CR treatment, AD mice showed improved ability of learning and spatial memory, and lower levels of Aβ40, Aβ42, IL-1β, TNF-α, and ROS in the brain. By sequencing 16S rDNA, we found that CR treatment resulted in significant diversity in composition and abundance of gut flora. At the phylum level, Deferribacteres (0.04%), Patescibacteria (0.14%), Tenericutes (0.03%), and Verrucomicrobia (0.5%) were significantly decreased in CR-treated AD mice; at the genus level, Dubosiella (10.04%), Faecalibaculum (0.04%), and Coriobacteriaceae UCG-002 (0.01%) were significantly increased in CR-treated AD mice by comparing with AL diet. Our results demonstrate that the attenuation of AD following CR treatment in APP/PS1 mice may result from alterations in the gut microbiome. Thus, gut flora could be a new target for AD prevention and therapy.

TaGSK3 regulates wheat development and stress adaptation through BR-dependent and BR-independent pathways.

The GSK3/SHAGGY-like kinase plays critical roles in plant development and response to stress, but its specific function remains largely unknown in wheat (Triticum aestivum L.). In this study, we investigated the function of TaGSK3, a GSK3/SHAGGY-like kinase, in wheat development and response to stress. Our findings demonstrated that TaGSK3 mutants had significant effects on wheat seedling development and brassinosteroid (BR) signalling. Quadruple and quintuple mutants showed amplified BR signalling, promoting seedling development, while a sextuple mutant displayed severe developmental defects but still responded to exogenous BR signals, indicating redundancy and non-BR-related functions of TaGSK3. A gain-of-function mutation in TaGSK3-3D disrupted BR signalling, resulting in compact and dwarf plant architecture. Notably, this mutation conferred significant drought and heat stress resistance of wheat, and enhanced heat tolerance independent of BR signalling, unlike knock-down mutants. Further research revealed that this mutation maintains a higher relative water content by regulating stomatal-mediated water loss and maintains a lower ROS level to reduces cell damage, enabling better growth under stress. Our study provides comprehensive insights into the role of TaGSK3 in wheat development, stress response, and BR signal transduction, offering potential for modifying TaGSK3 to improve agronomic traits and enhance stress resistance in wheat.

Icariin ameliorates osteoporosis in ovariectomized rats by targeting Cullin 3/Nrf2/OH pathway for osteoclast inhibition

Osteoporosis, characterized by low bone mass and bone microarchitecture breakdown, has become a growing public health problem. The increase in oxidative stress could lead to an imbalance between osteoblasts-mediated osteogenesis and osteoclast-mediated bone resorption, which gives rise to osteoporosis. Nrf2 is a master transcription factor that regulates oxidative stress and has recently been reported to take part in the development of osteoporosis. Icariin, a leading active flavonoid in herbal Epimedium pubescens, has significant antioxidant activity in and is widely applied for treating bone diseases. In this study, we aimed to explore the effect of icariin on osteoclastogenesis and its potential mechanism from the perspective of oxidative stress inhibition, using ovariectomized (OVX) rats and RANKL-induced RAW264.7 cells. Our results demonstrated that icariin-treated OVX rats exhibited higher bone density, fewer tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and lower ROS levels in bone tissues than vehicle-treated OVX rats. Also, icariin suppressed osteoclast differentiation and inhibited the expression of osteoclastogenesis-related genes, such as NFATc1, Ctsk, Trap, and c-Fos, in RANKL-induced RAW264.7 cells. Icariin also reduced intracellular ROS levels by increasing the expression of nuclear Nrf2 and HO-1. Further mechanistic studies showed icariin inhibited Cullin 3 expression and could delay Nrf2 degradation by reducing the ubiquitination of endogenous Nrf2 in RANKL-stimulated RAW264.7 cells, and these effects were markedly reversed by cullin three overexpression. These findings suggest icariin alleviated osteoporosis by suppressing osteoclastogenesis via targeting the Cullin 3/Nrf2/OH signaling pathway. Our study implied that icariin may be a potential candidate to treat osteoporosis.

Adaptive laboratory evolution of Naematelia aurantialba under high temperature for efficient production of exopolysaccharide

Liquid fermentation could revolutionize mushroom polysaccharide production, but the low temperature constraint hampers the process. This study implemented adaptive laboratory evolution (ALE) to enhance the thermotolerance of Naematelia aurantialba strains and increase expolysaccharide production. After 75 ALE cycles at 30 °C, the adaptive strain surpassed the wild-type strain by 5 °C. In a 7.5 L fermentor at 30 °C, the ALE strain yielded 17 % more exopolysaccharide than the wild type strain at 25 °C. Although the exopolysaccharide synthesized by both strains shares a consistent monosaccharide composition, infrared spectrum, and glycosidic bond composition, the ALE strain's exopolysaccharide has a larger molecular weight. Furthermore, the ALE strain's exopolysaccharide exhibits superior cryoprotection performance compared to that produced by the original strain. The adapted strain demonstrated lower ROS levels and increased activity of antioxidant enzymes, indicating improved performance. Fatty acid profiling and transcriptomics revealed reconfiguration of carbohydrate metabolism, amino acid metabolism, and membrane lipid synthesis in thermophilic strains, maintaining cellular homeostasis and productivity. This study provides efficient strains and fermentation methods for high-temperature mushroom polysaccharide production, reducing energy consumption and costs.

Hematopoiesis Revolves Around the Primordial Evolutional Rhythm of Purinergic Signaling and Innate Immunity – A Journey to the Developmental Roots

A cell's most significant existential task is to survive by ensuring proper metabolism, avoiding harmful stimuli, and adapting to changing environments. It explains why early evolutionary primordial signals and pathways remained active and regulate cell and tissue integrity. This requires energy supply and a balanced redox state. To meet these requirements, the universal intracellular energy transporter purine nucleotide-adenosine triphosphate (ATP) became an important signaling molecule and precursor of purinergic signaling after being released into extracellular space. Similarly, ancient proteins involved in intracellular metabolism gave rise to the third protein component (C3) of the complement cascade (ComC), a soluble arm of innate immunity. These pathways induce cytosol reactive oxygen (ROS) and reactive nitrogen species (RNS) that regulate the redox state of the cells. While low levels of ROS and RNS promote cell growth and differentiation, supra-physiological concentrations can lead to cell damage by pyroptosis. This balance explains the impact of purinergic signaling and innate immunity on cell metabolism, organogenesis, and tissue development. Subsequently, along with evolution, new regulatory cues emerge in the form of growth factors, cytokines, chemokines, and bioactive lipids. However, their expression is still modulated by both primordial signaling pathways. This review will focus on the data that purinergic signaling and innate immunity carry on their ancient developmental task in hematopoiesis and specification of hematopoietic stem/progenitor cells (HSPCs). Moreover, recent evidence shows both these regulatory pathways operate in a paracrine manner and inside HSPCs at the autocrine level.Graphical

Petal abscission is promoted by jasmonic acid-induced autophagy at Arabidopsis petal bases

In angiosperms, the transition from floral-organ maintenance to abscission determines reproductive success and seed dispersion. For petal abscission, cell-fate decisions specifically at the petal-cell base are more important than organ-level senescence or cell death in petals. However, how this transition is regulated remains unclear. Here, we identify a jasmonic acid (JA)-regulated chromatin-state switch at the base of Arabidopsis petals that directs local cell-fate determination via autophagy. During petal maintenance, co-repressors of JA signaling accumulate at the base of petals to block MYC activity, leading to lower levels of ROS. JA acts as an airborne signaling molecule transmitted from stamens to petals, accumulating primarily in petal bases to trigger chromatin remodeling. This allows MYC transcription factors to promote chromatin accessibility for downstream targets, including NAC DOMAIN-CONTAINING PROTEIN102 (ANAC102). ANAC102 accumulates specifically at the petal base prior to abscission and triggers ROS accumulation and cell death via AUTOPHAGY-RELATED GENEs induction. Developmentally induced autophagy at the petal base causes maturation, vacuolar delivery, and breakdown of autophagosomes for terminal cell differentiation. Dynamic changes in vesicles and cytoplasmic components in the vacuole occur in many plants, suggesting JA–NAC-mediated local cell-fate determination by autophagy may be conserved in angiosperms.

EMATOPOIESIS REVOLVES AROUND THE PRIMORDIAL EVOLUTIONAL RHYTHM OF INNATE IMMUNITY AND PURINERGIC SIGNALING – A JOURNEY TO THE DEVELOPMENTAL ROOTS

A cell’s most significant existential task is to survive by ensuring proper metabolism, avoiding harmful stimuli, and adapting to changing environments. It explains why early evolutionary primordial signals and pathways remained active and regulate cell and tissue integrity. This requires energy supply and a balanced redox state. To meet these requirements, ancient proteins involved in intracellular metabolism gave rise to the third protein component (C3) of the complement cascade (ComC), a soluble arm of innate immunity. Similarly, the universal intracellular energy transporter purine nucleotide-adenosine triphosphate (ATP) became an important signaling molecule and precursor of purinergic signaling after being released into extracellular space. These pathways induce cytosol reactive oxygen (ROS) and reactive nitrogen species (RNS) that regulate the redox state of the cells. While low levels of ROS and RNS promote cell growth and differentiation, supra-physi- ological concentrations can lead to cell damage by pyroptosis. This balance explains the impact of innate immunity and purinergic signaling on cell metabolism, organogenesis, and tissue development. Subsequently, along with evolution, new regulatory cues emerge in the form of growth factors, cytokines, chemokines, and bioactive lipids. However, their expression is still modulated by both primordial signaling pathways. Therefore, innate immunity and purinergic signaling carry on their ancient developmental task in hematopoiesis and hematopoietic stem/progenitor cells (HSPCs) specification. Recent evidence from my laboratory shows these regulatory pathways regulate hematopoiesis and operate both in a paracrine manner and at the autocrine level inside HSPCs.

Atrial natriuretic peptide protects against gut barrier injury through PLC-γ1/ROS feedback loop in rats following traumatic hemorrhagic shock

Introduction The mechanisms underlying the protective effects of atrial natriuretic peptide (ANP) on the gut barrier during traumatic hemorrhagic shock (THS) remain elusive. This study aimed to explore the potential role of ANP in safeguarding against gut barrier dysfunction after THS, focusing on the PLC-γ1/ROS feedback loop. Methods In our THS rat model, we randomly allocated male Sprague-Dawley rats to receive intravenous ANP with or without a concurrent NADPH oxidase/p38 MAPK inhibitor during the shock phase. After 24 h, we assessed circulatory and jejunal ANP, ROS, intestinal tight junction proteins, and apoptosis to evaluate the effects of ANP on the gut barrier and its interplay with intestinal ANP and ROS. Rat small intestinal epithelial cells (IECs) were also treated with ANP and subjected to hypoxia/re-oxygenation injury, with or without PI3K/PLC inhibition, to elucidate the relationship between ANP/ROS signaling and PLC-γ1. Furthermore, we modulated PLC-γ1 expression in these IECs to examine its impact on ROS and ANP production. Results Intravenous ANP administration at 0.025 μg/kg/min during THS significantly increased intestinal ANP and ROS levels at 24 h. ANP treatment enhanced the expression of intestinal tight junction proteins and reduced IEC apoptosis. Inhibition of circulatory ROS diminished intestinal ANP levels, while suppression of circulatory ANP led to a reduction in intestinal ROS. Decreasing PLC-γ1 expression in hypoxia/re-oxygenation-treated IECs resulted in lower ROS and ANP levels, whereas augmenting PLC-γ1 expression did not alter these levels. Additionally, PI3K inhibition markedly decreased PLC-γ1 expression in these cells. Conclusion ANP-induced protection of the intestinal barrier in THS is mediated by an intrinsic PLC-γ1/ROS positive feedback loop. ANP preserves gut barrier integrity and reduces IEC apoptosis through this mechanism. Further studies are warranted to investigate the interaction between IECs and other cellular components within the PLC-γ1/ROS loop.

Mitochondrial dysfunction and metabolic reprogramming induce macrophage pro-inflammatory phenotype switch and atherosclerosis progression in aging.

Aging increases the risk of atherosclerotic vascular disease and its complications. Macrophages are pivotal in the pathogenesis of vascular aging, driving inflammation and atherosclerosis progression. NOX4 (NADPH oxidase 4) expression increases with age, correlating with mitochondrial dysfunction, inflammation, and atherosclerosis. We hypothesized that the NOX4-dependent mitochondrial oxidative stress promotes aging-associated atherosclerosis progression by causing metabolic dysfunction and inflammatory phenotype switch in macrophages. We studied atherosclerotic lesion morphology and macrophage phenotype in young (5-month-old) and aged (16-month-old) Nox4 -/-/Apoe -/- and Apoe -/- mice fed Western diet. Young Nox4-/-/Apoe-/- and Apoe-/- mice had comparable aortic and brachiocephalic artery atherosclerotic lesion cross-sectional areas. Aged mice showed significantly increased lesion area compared with young mice. Aged Nox4-/-/Apoe-/- had significantly lower lesion areas than Apoe-/- mice. Compared with Apoe-/- mice, atherosclerotic lesions in aged Nox4-/-/Apoe-/- showed reduced cellular and mitochondrial ROS and oxidative DNA damage, lower necrotic core area, higher collagen content, and decreased inflammatory cytokine expression. Immunofluorescence and flow cytometry analysis revealed that aged Apoe-/- mice had a higher percentage of classically activated pro-inflammatory macrophages (CD38+CD80+) in the lesions. Aged Nox4-/-/Apoe-/- mice had a significantly higher proportion of alternatively activated pro-resolving macrophages (EGR2+/CD163+CD206+) in the lesions, with an increased CD38+/EGR2+ cell ratio compared with Apoe-/- mice. Mitochondrial respiration assessment revealed impaired oxidative phosphorylation and increased glycolytic ATP production in macrophages from aged Apoe-/- mice. In contrast, macrophages from Nox4-/-/Apoe-/- mice were less glycolytic and more aerobic, with preserved basal and maximal respiration and mitochondrial ATP production. Macrophages from Nox4-/-/Apoe-/- mice also had lower mitochondrial ROS levels and reduced IL1β secretion; flow cytometry analysis showed fewer CD38+ cells after IFNγ+LPS treatment and more EGR2+ cells after IL4 treatment than in Apoe-/- macrophages. In aged Apoe-/- mice, inhibition of NOX4 activity using GKT137831 significantly reduced macrophage mitochondrial ROS and improved mitochondrial function, resulting in decreased CD68+CD80+ and increased CD163+CD206+ lesion macrophage proportion and attenuated atherosclerosis. Our findings suggest that increased NOX4 in aging drives macrophage mitochondrial dysfunction, glycolytic metabolic switch, and pro-inflammatory phenotype, advancing atherosclerosis. Inhibiting NOX4 or mitochondrial dysfunction could alleviate vascular inflammation and atherosclerosis, preserving plaque integrity.

Local Oxygen Dictates Hematopoietic Cell Growth and Function

Functional hematopoietic stem (HSC) and progenitor cells (HPC) are responsible for the replenishment of blood/immune cells, are a source of treatment for diseases, and when dysregulated are a source of hematological neoplasia. To identify factors that impact HSC/HPC functional competency, we performed a transcriptomic screen which revealed that human cord blood (CB) HSCs/HPCs capable of early engraftment in an NSG mouse model of transplantation exhibited enriched expression of gene programs that vary with changes in oxygen (O 2) availability compared to non-transplanted input cells. This is physiologically and clinically relevant as HSCs/HPCs and diseased hematopoietic cells such as acute myeloid leukemia (AML) are exposed to O 2 tensions ranging from &amp;lt;1% to 21% as they function in various anatomical sites or are harvested for clinical applications. Thus, there are translational and practical rationales for understanding the growth and functional properties that hematopoietic cells exhibit across the range of O 2 they experience physiologically or artificially, which we examined here. First we found significantly different frequencies of HSCs/HPCs and colony forming units (CFU) in human bone marrow (BM), mobilized peripheral blood (PB), and CB, which have varied O 2 tensions, suggesting O 2 availability may impact hematopoiesis. To directly examine this, human CB CD34+ cells were split and plated in CFU assays or expanded in growth stimulating conditions at 1% O 2, 3% O 2, 5% O 2, 14% O 2 (physiologic tensions), or 21% O 2 (processing tension) . HPCs expanded best and formed more differentiated CFUs in high physiologic O 2 tensions and extra physiologic O 2 tensions, while HSCs expanded better and more potent CFUs were formed in a mid-physiologic O 2 tension (5%). This suggests that O 2 dependent pathways are critical to HSC/HPC growth but there may be a “sweet spot” for early HSCs/HPCs that allows for proliferation while maintaining self-renewal and potency to maintain a pool of primitive cells, possibly mirroring in vivo O 2 responses. In in vivo functional HSC/HPC analyses, NSG mice transplanted with CB units expanded in physiologic O 2 exhibited significantly better early neutrophil recovery and long-term myeloid reconstitution compared to input, suggesting that growth and maintenance of HSC/ myeloid HPC functional competency is better balanced in physiologic O 2 compared to extra physiologic tensions. Platelet recovery and lymphoid reconstitution were significantly better in mice receiving uncultured control CB, suggesting expansion does not affect all HSC/HPC subpopulations equally. Mechanistically, cells grown in low O 2 tensions (1-3%) had higher frequencies of early HSCs/HPCs, significantly lower intracellular ROS levels, a significantly higher percentage of cells in cell cycle G0, and lower levels of phosphorylated γH2AX than those grown in higher O 2. These data suggest that while to some extent HSCs/HPCs require O 2 to proliferate, they also exhibit properties associated with exhaustion when maintained in higher O 2. We next examined the effects varying O 2 tensions have on diseased hematopoietic cells using primary mouse AML cells or oncogene transformed human CB CD34+ cell lines. AML cells proliferated significantly more rapidly and formed more CFUs in higher O 2 tensions, suggesting that O 2 sensing pathways may be a dependency in AML that could affect disease progression as local O 2 tensions become perturbed. AML cells grown in lower O 2 exhibited lower resting levels of phosphorylated γH2AX (measured by flow cytometry) and lower O 2 consumption rates (measured by Seahorse metabolic flux analyzer), suggesting they are less susceptible to cellular stress. In line with this, AML cells maintained in low O 2 exhibited significantly better proliferation and recovery after treatment with the chemotherapeutic cytarabine compared to those in higher O 2 tensions. Thus, diseased cells growing in lower O 2 niches may be less susceptible to treatment and therefore may be a source of chemoresistance or relapse. These data reveal important roles for O 2 in balancing hematopoietic cell growth with normal and diseased cell function. Manipulating O 2 tension or O 2 sensing pathways could be used to improve HSC/HPC expansion for transplantation or to treat disease. Transcriptomic studies of HSCs/HPCs and AML cells are ongoing to determine the O 2 sensing mechanisms that dictate cellular function.

Overexpression of IlHMA2, from Iris lactea, Improves the Accumulation of and Tolerance to Cadmium in Tobacco.

Long-distance transport cadmium (Cd) from roots to shoots is a key factor for Cd phytoremediation. Our previous study indicated that heavy metal P1B2-ATPases, IlHMA2, was involved in improving the accumulation of Cd via mediated long-distance transport Cd, contributing to the phytoremediation in Cd accumulator Iris lactea. However, whether the overexpression of IlHMA2 could enhance the accumulation and tolerance to Cd remains unclear in plants. Here, we generated transgenic tobacco overexpressing IlHMA2 and tested its effect on the translocation and accumulation of Cd and zinc (Zn), as well as the physio-biochemical characteristics under 50 mg/L Cd exposure. The overexpression of IlHMA2 significantly increased Cd concentrations in xylem saps, resulting in enhanced root-to-shoot Cd translocation compared with wild-type. Meanwhile, overexpressing IlHMA2 promoted Zn accumulations, accompanied by elevating proline contents and antioxidant enzyme activity (SOD, POD, and CAT) to diminish the overproduction of ROS in transgenic tobacco. These pieces of evidence suggested that higher Zn concentrations and lower ROS levels could tremendously alleviate Cd toxicity for transgenic tobacco, thereby improving the growth and tolerance. Overall, the overexpression of IlHMA2 could facilitate Cd accumulation and enhance its tolerance in tobacco exposed to Cd contaminations. This would provide a valuable reference for improving Cd phytoremediation efficiency.

MsDjB4, a HSP40 Chaperone in Alfalfa (Medicago sativa L.), Improves Alfalfa Hairy Root Tolerance to Aluminum Stress.

The toxicity of aluminum (Al) in acidic soils poses a significant limitation to crop productivity. In this study, we found a notable increase in DnaJ (HSP40) expression in the roots of Al-tolerant alfalfa (WL-525HQ), which we named MsDjB4. Transient conversion assays of tobacco leaf epidermal cells showed that MsDjB4 was targeted to the membrane system including Endoplasmic Reticulum (ER), Golgi, and plasma membrane. We overexpressed (MsDjB4-OE) and suppressed (MsDjB4-RNAi) MsDjB4 in alfalfa hairy roots and found that MsDjB4-OE lines exhibited significantly better tolerance to Al stress compared to wild-type and RNAi hairy roots. Specifically, MsDjB4-OE lines had longer root length, more lateral roots, and lower Al content compared to wild-type and RNAi lines. Furthermore, MsDjB4-OE lines showed lower levels of lipid peroxidation and ROS, as well as higher activity of antioxidant enzymes SOD, CAT, and POD compared to wild-type and RNAi lines under Al stress. Moreover, MsDjB4-OE lines had higher soluble protein content compared to wild-type and RNAi lines after Al treatment. These findings provide evidence that MsDjB4 contributes to the improved tolerance of alfalfa to Al stress by facilitating protein synthesis and enhancing antioxidant capacity.

Protective effects of alpha lipoic acid (ALA) are mediated by hormetic mechanisms.

The endogenous and dietary agent, alpha lipoic acid (ALA), is evaluated for its capacity to induce a broad spectrum of adaptive responses via hormetic dose responses and their underlying mechanisms. ALA was shown to induce hormetic effects in a wide range of experimental models within in vitro and in vivo experimental settings which included direct exposure and pre- and post-conditioning experimental protocols. The hormetic effects occur in a broad range of organ systems, including the brain, heart, kidney and other tissues, with possible public health and clinical/therapeutic applications linked to reducing the onset and progression of neurogenerative diseases and also in the preservation of sperm health and functionality during cryopreservation. This paper provides the first integrated assessment of ALA-induced hormetic dose responses. Underlying mechanisms that mediated the occurrence of ALA-induced hormetic effects involved the induction of low levels of ROS that activate key cell signaling antioxidant (e.g. Nrf2) pathways.