Melatonin-Mediated Suppression of mtROS-JNK-FOXO1 Pathway Alleviates Hypoxia-Induced Apoptosis in Porcine Granulosa Cells.

Numerous studies have established that the hypoxic conditions within ovarian follicles induce apoptosis in granulosa cells (GCs), a pivotal hallmark of follicular atresia. Melatonin (N-acetyl-5-methoxytryptamine, MT), a versatile antioxidant naturally present in follicular fluid, acts as a safeguard for maintaining GCs' survival during stress exposure. In this study, we unveil an innovative protective mechanism of melatonin against hypoxia-triggered GC apoptosis by selectively inhibiting mitochondrial ROS (mtROS) generation. Specifically, under hypoxic conditions, a gradual accumulation of mitochondrial ROS occurred, consequently activating the JNK-FOXO1 pathway, and driving GCs toward apoptosis. The blocking of JNK or FOXO1 diminished hypoxia-induced GC apoptosis, but this effect was nullified in the presence of GSH, indicating that mtROS instigates apoptosis through the JNK-FOXO1 pathway. Consistent with this, hypoxic GCs treated with melatonin exhibited decreased levels of mtROS, reduced JNK-FOXO1 activation, and mitigated apoptosis. However, the protective capabilities of melatonin were attenuated upon inhibiting its receptor MTNR1B, accompanied by the decreased expression of antioxidant genes. Notably, SOD2, a key mitochondrial antioxidant gene modulated by the melatonin-MTNR1B axis, effectively inhibited the activation of mtROS-JNK-FOXO1 and subsequent apoptosis, whereas SOD2 knockdown abrogated the protective role of melatonin in hypoxic GCs. In conclusion, our study elucidates that melatonin, through MTNR1B activation, fosters SOD2 expression, effectively quelling mtROS-JNK-FOXO1-mediated apoptosis in follicular GCs under hypoxic stress.

Global Transcriptome and Weighted Gene Co-expression Network Analyses of Growth-Stage-Specific Drought Stress Responses in Maize.

Drought is the major abiotic stress threatening maize (Zea mays L.) production globally. Despite recent scientific headway in deciphering maize drought stress responses, the overall picture of key genes, pathways, and co-expression networks regulating maize drought tolerance is still fragmented. Therefore, deciphering the molecular basis of maize drought tolerance remains pertinent. Here, through a comprehensive comparative leaf transcriptome analysis of drought-tolerant hybrid ND476 plants subjected to water-sufficient and water-deficit treatment conditions at flared (V12), tasseling (VT), the prophase of grain filling (R2), and the anaphase of grain filling (R4) crop growth stages, we report growth-stage-specific molecular mechanisms regulating maize drought stress responses. Based on the transcriptome analysis, a total of 3,451 differentially expressed genes (DEGs) were identified from the four experimental comparisons, with 2,403, 650, 397, and 313 DEGs observed at the V12, VT, R1, and R4 stages, respectively. Subsequently, 3,451 DEGs were divided into 12 modules by weighted gene co-expression network analysis (WGCNA), comprising 277 hub genes. Interestingly, the co-expressed genes that clustered into similar modules exhibited diverse expression tendencies and got annotated to different GO terms at different stages. MapMan analysis revealed that DEGs related to stress signal transduction, detoxification, transcription factor regulation, hormone signaling, and secondary metabolites biosynthesis were universal across the four growth stages. However, DEGs associated with photosynthesis and amino acid metabolism; protein degradation; transport; and RNA transcriptional regulation were uniquely enriched at the V12, VT, R2, and R4 stages, respectively. Our results affirmed that maize drought stress adaptation is a growth-stage-specific response process, and aid in clarifying the fundamental growth-stage-specific mechanisms regulating drought stress responses in maize. Moreover, genes and metabolic pathways identified here can serve as valuable genetic resources or selection targets for further functional validation experiments.

Superselective Arterial Hyaluronidase Thrombolysis Is Not an Effective Treatment for Hyaluronic Acid-Induced Retinal Artery Occlusion: Study in a Rabbit Model.

BACKGROUND: Worldwide application of hyaluronic acid has brought about severe complications, including central retinal arterial occlusion, which leads to a deleterious effect on vision. The current study explored the efficacy of superselective arterial hyaluronidase thrombolysis in rabbit retinal artery occlusion induced by hyaluronic acid. METHODS: Occlusion of the internal/external ophthalmic artery in New Zealand White rabbits was induced with superselective injection of hyaluronic acid. Superselective subtraction angiography and fundus examination were conducted to confirm and evaluate the artery embolism. After 30 minutes of embolism, hyaluronidase was injected in the occluded artery through superselective arterial intubation. RESULTS: Compared with preoperative and contralateral eyes, the postoperative eyes showed the symptoms of central retinal arterial occlusion and embolization, confirmed by digital subtraction angiography. After intraarterial hyaluronidase thrombolysis, the embolization failed to dissolve as shown on funduscopic and angiographic examinations. CONCLUSIONS: Superselective ophthalmic artery intervention could accurately and successfully establish the animal models of retinal artery occlusion induced by hyaluronic acid. The precise occlusion site of the retinal artery and complete embolism were confirmed by ophthalmologic examinations. Intraarterial hyaluronidase thrombolysis might not be an effective method to treat retinal artery occlusion induced by hyaluronic acid.

Comparative transcriptomic and physiological analyses of contrasting hybrid cultivars ND476 and ZX978 identify important differentially expressed genes and pathways regulating drought stress tolerance in maize.

BACKGROUND: Drought is the major abiotic stress factor that negatively influences growth and yield in cereal grain crops such as maize (Zea mays L.). A multitude of genes and pathways tightly modulate plant growth, development and responses to environmental stresses including drought. Therefore, crop breeding efforts for enhanced drought resistance require improved knowledge of plant drought responses. OBJECTIVE: Here, we sought to elucidate the molecular and physiological mechanisms underpinning maize drought stress tolerance. METHODS: We therefore applied a 12-day water-deficit stress treatment to maize plants of two contrasting (drought tolerant ND476 and drought sensitive ZX978) hybrid cultivars at the late vegetative (V12) growth stage and performed a large-scale RNA sequencing (RNA-seq) transcriptome analysis of the leaf tissues. RESULTS: A comparative analysis of the two genotypes leaf transcriptomes and physiological parameters revealed the key differentially expressed genes (DEGs) and metabolic pathways that respond to drought in a genotype-specific manner. A total of 3114 DEGs were identified, with 21 DEGs being specifically expressed in tolerant genotype ND476 in response to drought stress. Of these, genes involved in secondary metabolites biosynthesis, transcription factor regulation, detoxification and stress defense were highly expressed in ND476. Physiological analysis results substantiated our RNA-seq data, with ND476 exhibiting better cell water retention, higher soluble protein content and guaiacol peroxidase activity, along with low lipid peroxidation extent than the sensitive cultivar ZX978 under drought conditions. CONCLUSION: Our findings enrich the maize genetic resources and enhance our further understanding of the molecular mechanisms regulating drought stress tolerance in maize. Additionally, the DEGs screened in this study may provide a foundational basis for our future targeted cloning studies.

Comparative Proteomic and Morpho-Physiological Analyses of Maize Wild-Type Vp16 and Mutant vp16 Germinating Seed Responses to PEG-Induced Drought Stress.

Drought stress is a major abiotic factor compromising plant cell physiological and molecular events, consequently limiting crop growth and productivity. Maize (Zea mays L.) is among the most drought-susceptible food crops. Therefore, understanding the mechanisms underlying drought-stress responses remains critical for crop improvement. To decipher the molecular mechanisms underpinning maize drought tolerance, here, we used a comparative morpho-physiological and proteomics analysis approach to monitor the changes in germinating seeds of two incongruent (drought-sensitive wild-type Vp16 and drought-tolerant mutant vp16) lines exposed to polyethylene-glycol-induced drought stress for seven days. Our physiological analysis showed that the tolerant line mutant vp16 exhibited better osmotic stress endurance owing to its improved reactive oxygen species scavenging competency and robust osmotic adjustment as a result of greater cell water retention and enhanced cell membrane stability. Proteomics analysis identified a total of 1200 proteins to be differentially accumulated under drought stress. These identified proteins were mainly involved in carbohydrate and energy metabolism, histone H2A-mediated epigenetic regulation, protein synthesis, signal transduction, redox homeostasis and stress-response processes; with carbon metabolism, pentose phosphate and glutathione metabolism pathways being prominent under stress conditions. Interestingly, significant congruence (R2 = 81.5%) between protein and transcript levels was observed by qRT-PCR validation experiments. Finally, we propose a hypothetical model for maize germinating-seed drought tolerance based on our key findings identified herein. Overall, our study offers insights into the overall mechanisms underpinning drought-stress tolerance and provides essential leads into further functional validation of the identified drought-responsive proteins in maize.

Comparative Proteomics and Physiological Analyses Reveal Important Maize Filling-Kernel Drought-Responsive Genes and Metabolic Pathways.

Despite recent scientific headway in deciphering maize (Zea mays L.) drought stress responses, the overall picture of key proteins and genes, pathways, and protein-protein interactions regulating maize filling-kernel drought tolerance is still fragmented. Yet, maize filling-kernel drought stress remains devastating and its study is critical for tolerance breeding. Here, through a comprehensive comparative proteomics analysis of filling-kernel proteomes of two contrasting (drought-tolerant YE8112 and drought-sensitive MO17) inbred lines, we report diverse but key molecular actors mediating drought tolerance in maize. Using isobaric tags for relative quantification approach, a total of 5175 differentially abundant proteins (DAPs) were identified from four experimental comparisons. By way of Venn diagram analysis, four critical sets of drought-responsive proteins were mined out and further analyzed by bioinformatics techniques. The YE8112-exclusive DAPs chiefly participated in pathways related to "protein processing in the endoplasmic reticulum" and "tryptophan metabolism", whereas MO17-exclusive DAPs were involved in "starch and sucrose metabolism" and "oxidative phosphorylation" pathways. Most notably, we report that YE8112 kernels were comparatively drought tolerant to MO17 kernels attributable to their redox post translational modifications and epigenetic regulation mechanisms, elevated expression of heat shock proteins, enriched energy metabolism and secondary metabolites biosynthesis, and up-regulated expression of seed storage proteins. Further, comparative physiological analysis and quantitative real time polymerase chain reaction results substantiated the proteomics findings. Our study presents an elaborate understanding of drought-responsive proteins and metabolic pathways mediating maize filling-kernel drought tolerance, and provides important candidate genes for subsequent functional validation.

Key Maize Drought-Responsive Genes and Pathways Revealed by Comparative Transcriptome and Physiological Analyses of Contrasting Inbred Lines.

To unravel the molecular mechanisms underpinning maize (Zea mays L.) drought stress tolerance, we conducted comprehensive comparative transcriptome and physiological analyses of drought-tolerant YE8112 and drought-sensitive MO17 inbred line seedlings that had been exposed to drought treatment for seven days. Resultantly, YE8112 seedlings maintained comparatively higher leaf relative water and proline contents, greatly increased peroxidase activity, but decreased malondialdehyde content, than MO17 seedlings. Using an RNA sequencing (RNA-seq)-based approach, we identified a total of 10,612 differentially expressed genes (DEGs). From these, we mined out four critical sets of drought responsive DEGs, including 80 specific to YE8112, 5140 shared between the two lines after drought treatment (SD_TD), five DEGs of YE8112 also regulated in SD_TD, and four overlapping DEGs between the two lines. Drought-stressed YE8112 DEGs were primarily associated with nitrogen metabolism and amino-acid biosynthesis pathways, whereas MO17 DEGs were enriched in the ribosome pathway. Additionally, our physiological analyses results were consistent with the predicted RNA-seq-based findings. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) analysis and the RNA-seq results of twenty representative DEGs were highly correlated (R² = 98.86%). Crucially, tolerant line YE8112 drought-responsive genes were predominantly implicated in stress signal transduction; cellular redox homeostasis maintenance; MYB, NAC, WRKY, and PLATZ transcriptional factor modulated; carbohydrate synthesis and cell-wall remodeling; amino acid biosynthesis; and protein ubiquitination processes. Our findings offer insights into the molecular networks mediating maize drought stress tolerance.