Comparative Transcriptome Analyses Reveal Conserved and Distinct Mechanisms of the SDHI Fungicide Benzovindiflupyr Inhibiting Colletotrichum

Colletotrichum leaf disease (CLD) is an annual production concern for commercial growers worldwide. The succinate dehydrogenase inhibitor (SDHI) fungicide benzovindiflupyr shows higher bioactivity against CLD than other SDHIs. However, the mechanism underlying such difference remains unclear. In this study, benzovindiflupyr exhibit good inhibitory activity against C. siamense and C. nymphaeae in vitro and in vivo. To reveal its mechanism for inhibiting Colletotrichum, we compared transcriptomes of C. siamense and C. nymphaeae under treatment with benzovindiflupyr and boscalid. Benzovindiflupyr exhibited higher inhibitory activity against SDH enzyme than boscalid, resulting in a greater reduction in the ATP content of Colletotrichum isolates. Most of the metabolic pathways induced in these fungicide-treated isolates were similar, indicating that benzovindiflupyr exhibited a conserved mechanism of SDHIs inhibiting Colletotrichum. At the same level of suppressive SDH activity, benzovindiflupyr activated more than three times greater gene numbers of Colletotrichum than boscalid, suggesting that benzovindiflupyr could activate distinct mechanisms against Colletotrichum. Especially, membrane-related gene ontology terms, mainly including intrinsic components of membrane, were highly abundant for the benzovindiflupyr-treated isolates rather than boscalid-treated isolates. Only benzovindiflupyr increased the relative conductivities of hyphae, indicating that it could damage the cell membrane and increase of mycelial electrolyte leakage. Thus, we proposed that the high bioactivity of benzovindiflupyr against Colletotrichum by inhibiting SDH activity and damaging the cell membrane at the same time. The research improves our understanding the mode of action of SDHI fungicides against Colletotrichum.

Transcriptomics of single dose and repeated carbon black and ozone inhalation co-exposure highlight progressive pulmonary mitochondrial dysfunction

Background Air pollution is a complex mixture of particles and gases, yet current regulations are based on single toxicant levels failing to consider potential interactive outcomes of co-exposures. We examined transcriptomic changes after inhalation co-exposure to a particulate and a gaseous component of air pollution and hypothesized that co-exposure would induce significantly greater impairments to mitochondrial bioenergetics. A whole-body inhalation exposure to ultrafine carbon black (CB), and ozone (O3) was performed, and the impact of single and multiple exposures was studied at relevant deposition levels. C57BL/6 mice were exposed to CB (10 mg/m3) and/or O3 (2 ppm) for 3 h (either a single exposure or four independent exposures). RNA was isolated from lungs and mRNA sequencing performed using the Illumina HiSeq. Lung pathology was evaluated by histology and immunohistochemistry. Electron transport chain (ETC) activities, electron flow, hydrogen peroxide production, and ATP content were assessed. Results Compared to individual exposure groups, co-exposure induced significantly greater neutrophils and protein levels in broncho-alveolar lavage fluid as well as a significant increase in mRNA expression of oxidative stress and inflammation related genes. Similarly, a significant increase in hydrogen peroxide production was observed after co-exposure. After single and four exposures, co-exposure revealed a greater number of differentially expressed genes (2251 and 4072, respectively). Of these genes, 1188 (single exposure) and 2061 (four exposures) were uniquely differentially expressed, with 35 mitochondrial ETC mRNA transcripts significantly impacted after four exposures. Both O3 and co-exposure treatment significantly reduced ETC maximal activity for complexes I (− 39.3% and − 36.2%, respectively) and IV (− 55.1% and − 57.1%, respectively). Only co-exposure reduced ATP Synthase activity (− 35.7%) and total ATP content (30%). Further, the ability for ATP Synthase to function is limited by reduced electron flow (− 25%) and translation of subunits, such as ATP5F1, following co-exposure. Conclusions CB and O3 co-exposure cause unique transcriptomic changes in the lungs that are characterized by functional deficits to mitochondrial bioenergetics. Alterations to ATP Synthase function and mitochondrial electron flow underly a pathological adaptation to lung injury induced by co-exposure.

Estrogen Regulates Glucose Metabolism in Cattle Neutrophils Through Autophagy

Hypoglycemia resulting from a negative energy balance (NEB) in periparturient cattle is the major reason for a reduced glycogen content in polymorphonuclear neutrophils (PMNs). The lack of glycogen induces PMNs dysfunction and is responsible for the high incidence of perinatal diseases. The perinatal period is accompanied by dramatic changes in sex hormones levels of which estrogen (17β-estradiol, E2) has been shown to be closely associated with PMNs function. However, the precise regulatory mechanism of E2 on glucose metabolism in cattle PMNs has not been elucidated. Cattle PMNs were cultured in RPMI 1640 with 2.5 (LG), 5.5 (NG) and 25 (HG) mM glucose and E2 at 20 (EL), 200 (EM) and 450 (EH) pg/mL. We found that E2 maintained PMNs viability in different glucose conditions, and promoted glycogen synthesis by inhibiting PFK1, G6PDH and GSK-3β activity in LG while enhancing PFK1 and G6PDH activity and inhibiting GSK-3β activity in HG. E2 increased the ATP content in LG but decreased it in HG. This indicated that the E2-induced increase/decrease of ATP content may be independent of glycolysis and the pentose phosphate pathway (PPP). Further analysis showed that E2 promoted the activity of hexokinase (HK) and GLUT1, GLUT4 and SGLT1 expression in LG, while inhibiting GLUT1, GLUT4 and SGLT1 expression in HG. Finally, we found that E2 increased LC3, ATG5 and Beclin1 expression, inhibited p62 expression, promoting AMPK-dependent autophagy in LG, but with the opposite effect in HG. Moreover, E2 increased the Bcl-2/Bax ratio and decreased the apoptosis rate of PMNs in LG but had the opposite effect in HG. These results showed that E2 could promote AMPK-dependent autophagy and inhibit apoptosis in response to glucose-deficient environments. This study elucidated the detailed mechanism by which E2 promotes glycogen storage through enhancing glucose uptake and retarding glycolysis and the PPP in LG. Autophagy is essential for providing ATP to maintain the survival and immune potential of PMNs. These results provided significant evidence for further understanding the effects of E2 on PMNs immune potential during the hypoglycemia accompanying perinatal NEB in cattle.

CPX-351, a Dual-Drug Liposomal Formulation, Alleviates the Cardiotoxicity of Daunorubicin and Cytarabine to Induced Human Pluripotent Stem Cell-Derived Cardiomyocytes

Introduction: CPX-351 (US: Vyxeos ®; EU: Vyxeos ® Liposomal), a dual-drug liposomal encapsulation of daunorubicin + cytarabine at a synergistic 1:5 molar ratio, is a standard of care for therapy-related acute myeloid leukemia (AML) or AML with myelodysplastic-related changes. Daunorubicin, an anthracycline, is a known cardiotoxicant. While liposomal formulations have shown promise in mitigating these effects (O'Brien, et al. Ann Oncol 2004), this potential advantage is difficult to assess clinically due to numerous confounding factors. Here, we sought to develop and employ an in vitro model to study the relative toxicity of CPX-351 versus free daunorubicin + cytarabine applied as a combination at the same concentrations. Model development: The hiPSC-derived cardiomyocytes used in this study were derived from fibroblasts obtained from a single adult Caucasian female donor with no known diseases, induced into a pluripotent state, and then reprogrammed into cardiomyocytes (CDI Datasheet 2018). They present many of the characteristics of healthy human cardiac muscle cells, including gene and protein expression and rhythmic beating. During model development, the prototypical compounds doxorubicin and liposomal doxorubicin were used as proof-of-concept to establish the translational value of the model because clinical data regarding their relative cardiotoxicity were available (O'Brien, et al. Ann Oncol 2004). The model recapitulated the cumulative toxicity of free doxorubicin and differentiated between liposomal and free drug. The in vitro model was then applied to compare the relative cardiotoxicity of CPX-351 versus the combination of free daunorubicin + cytarabine (1:5), which were applied to the cardiomyocytes for 24 hours on Days 1, 3, and 5 at concentrations ranging from 0 to 1,000 ng/mL daunorubicin (0 to 2,273 ng/mL cytarabine). Bright-field microscopy imaging, lactate dehydrogenase (LDH) release (reflects cell membrane integrity), ATP content (indicates metabolic activity), cell beat rate (indicates cardiomyocyte function), and cardiac biomarkers (FABP3, cardiac troponin I, NT-proBNP, and BNP) were evaluated on Days 2, 4, 6, and 8. Results: Qualitative review of the microscopic images suggested single dose- and cumulative dose-dependent cytotoxicity of free daunorubicin + cytarabine, especially at the highest doses on Day 8. These observations were confirmed by dose-dependent increases in single-day or cumulative LDH activity in the cell media and decreases in ATP content starting on Day 4. Specifically, after a single 24-hour exposure, LDH activity (a measure of plasma membrane damage) was comparable to that of saline-treated cardiomyocytes for both the free-drug combination and CPX-351. However, after repeated exposure to the free-drug combination, increasing drug concentration was associated with increasing LDH activity in the media, peaking at levels ~12 times the saline control on Day 8. In contrast, the LDH activity following repeated equivalent doses of CPX-351 was only ~3 times the saline control. Conversely, ATP content (a measure of cellular metabolic fitness) gradually decreased between Day 2 and Day 8. The ATP depletion (Day 8 vs Day 2) was more profound in cardiomyocytes exposed to the free-drug combination (−98.1%) than in those exposed to equivalent concentrations of CPX-351 (−38.5%). Following repeated exposure to the free-drug combination, the cell beat rate demonstrated a biphasic response consisting of an initial increase followed by a significant slowing and sometimes arrest of beating, demonstrating significant injury; this effect was not observed following repeated exposure to CPX-351. Finally, the cardiac biomarkers FABP3 and cardiac troponin I were significantly released from cardiomyocytes exposed to the free-drug combination, but not from those exposed to CPX-351, even after 3 repeated exposures. Conclusions: Overall, at equivalent concentrations administered on the same schedule, CPX-351 was considerably less toxic to hiPSC-derived cardiomyocytes than the free-drug combination of daunorubicin + cytarabine, as measured by viability (imaging, LDH release), metabolic health (ATP content), function (beating rate), and the cardiac biomarkers FABP3 and cardiac troponin I. Clinical data are needed to confirm the reduced cardiotoxicity observed with CPX-351 versus free drugs in this in vitro model. Disclosures Fortin: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. LaCroix: StemoniX: Current Employment; Jazz Pharmaceuticals: Consultancy. Wang: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Cheung: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

Hydrogen-Rich Medium Ameliorates Lipopolysaccharides-Induced Mitochondrial Fission and Dysfunction in Human Umbilical Vein Endothelial Cells (Huvecs) via Up-Regulating HO-1 Expression

Background Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. It has been showed that the change of mitochondrial dynamics has been proved to be one of the main causes of death in patients with severe sepsis. And hydrogen has been proved to exert its protective effects against sepsis via heme oxygenase-1 (HO-1). This study was designed to demonstrate that whether the benefit effects of hydrogen can maintain the dynamic process of mitochondrial fusion/fission to mitigate human umbilical vein endothelial cells (HUVECs) injury exposed to endotoxin through HO-1. Methods HUVECs cells cultured with medium which contained Lipopolysaccharides (LPS), Saline, hydrogen, Mdivi-1 (a dynamin-related protein 1 [Drp1] inhibitor) or zinc protoporphyrin Ⅸ (Znpp) (a HO-1 inhibitor) were also used in the research. Cell death and apoptosis were assessed using FITC annexin V and PI. Mitochondria were stained with Mitotracker orange and observed by confocal microscope. Oxygen consumption rate was assessed by seahorse xf24 extracellular analyzer. Mitochondrial membrane potential monitored by JC-1 dye. The expressions of Drp1 and HO-1 were tested by Western blot. The co-localization of Drp1 and mitochondria was determined by immunofluorescence. Results LPS caused a decrease in ATP content, mitochondrial membrane potential, and maximal respiration rate. At the same time, increased expression of Drp1 were observed in LPS-stimulated HUVECs, concomitantly with excessive mitochondrial fission. We found that hydrogen-rich medium can increase ATP content, mitochondrial membrane potential and maximal respiration rate, and decrease the expression of Drp1 in LPS-treated HUVECs. Meanwhile, hydrogen can ameliorate excessive mitochondrial fission caused by LPS. Furthermore, hydrogen-rich medium had a similar effect to Mdivi-1, a mitochondrial fission blocker. Both of them rescued the up-regulation of Drp1 and mitochondrial fission induced by LPS, then normalized mitochondrial shape after LPS stimulation. But after Znpp pretreatment, HO-1 expression was inhibited and the protective effects of hydrogen were abrogated. Conclusions Hydrogen-rich medium can alleviate the LPS-induced mitochondrial fusion/fission and dysfunction in HUVECs via HO-1 up-regulation.

XinJiaCongRongTuSiZiWan Protects TP-Induced Rats from Oxidative Stress Injury via Mitophagy Mediated PINK1/ Parkin Signaling Pathway

Background: Oxidative stress is one of main molecular mechanisms involved in toxicity of triptolide (TP). Although our group has discovered the effectiveness of XinJiaCongRongTuSiZiWan (XJCRTSZW) on premature ovarian failure (POF) and polycystic ovary syndrome (PCOS), whether the protective role of XJCRTSZW being associated with oxidative stress is still totally understood. Methods: Adult female Sprague-Dawley rats and human ovarian granulosa cell lines were treated with TP, and then treated with XinJiaCongRongTuSiZiWan (XJCRTSZW). Histological analysis and follicle count were executed using H&E staining. Hormone (E2, AMH, P, FSH and LH) concentrations, oxidative stress indicators (SOD and MDA), apoptosis rate, ATP content, mitochondrial membrane potential (MMP), cell viability, mitophagy and relative mRNA and protein levels (LC3-Ⅱ/LC3-Ⅰ, p62, Hsp60, PINK1 and Parkin) were detected by ELISA, commercial biochemical detection kits, flow cytometry, JC-1 staining, CCK-8, transmission electron microscope and western blotting respectively. Results: XJCRTSZW treatment observably ameliorated the TP-induced the pathological symptoms, including the decreased primordial follicles, primary follicles and secondary follicles numbers in the cortical area, the increased numbers of atretic follicles, necrotic and shedding, and nuclear constriction and collapse with cystic dilatation in vivo. Furthermore, XJCRTSZW treatment observably enhanced the TP-induced reduction of E2, AMH and P concentrations, SOD concentrations, ATP content, MMP, p62 and Hsp60 mRNA and protein level, but, diminished the TP-induced elevation of FSH and LH concentrations, MDA level, ROS level, apoptosis rate, mitophagy, and the mRNA and protein expression of LC3-Ⅱ/LC3-Ⅰ, PINK1 and Parkin both in vivo and in vitro. In addition, XJCRTSZW treatment markedly increased the TP-induced reduction of cell viability in vitro.Conclusion: XinJiaCongRongTuSiZiWan protects TP-induced rats from oxidative stress injury via mitophagy mediated PINK1/ Parkin signaling pathway.

Effects of Bushen-Tiaojing-Fang on the pregnancy outcomes of infertile patients with repeated controlled ovarian stimulation

Bushen-Tiaojing-Fang (BSTJF) is commonly used to treat infertility. This study investigated the effects of BSTJF on the pregnancy outcomes of patients with repeated controlled ovarian stimulation (COS), on mitochondrial function, and on oxidative stress in ovarian granulosa cells (GCs) and follicular fluid (FF). The samples and clinical data of 97 patients, including 35 in the control group, 29 in the placebo group and 33 in the BSTJF group, were collected for this study. The mitochondrial ultrastructure, ATP content, mitochondrial DNA (mtDNA) number, 8-hydroxy-2-deoxyguanosine (8-OHdG), Mn-superoxide dismutase (Mn-SOD), glutathione peroxidase (GSH-Px) activity levels, and mRNA expression levels of Mn-SOD, GSH-Px, and nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) were analyzed. The high-grade embryo (P < 0.001), implantation (P = 0.033), and clinical pregnancy (P = 0.031) rates, as well as the ATP content (P = 0.014), mtDNA number (P = 0.035), GSH-Px activity (P = 0.004 in GCs and P = 0.008 in FF) and mRNA expression levels (P = 0.019), were significantly lower in the placebo group than in the control group, whereas the 8-OHdG content was significantly (P = 0.006 in FF) higher in the placebo group than in the control group. Compared with those in the placebo group, the high-grade embryo rate (P = 0.007), antioxidant enzyme activity (P = 0.037 and 0.036 in Mn-SOD; P = 0.047 and 0.030 in GSH-Px) and mRNA level (P < 0.001 in Nrf2, P = 0.039 in Mn-SOD and P = 0.002 in GSH-Px) were significantly higher in the BSTJF group, as were changes in mitochondrial ultrastructure, ATP (P = 0.040) and mtDNA number (P = 0.013). In conclusion, BSTJF can improve oxidative stress in patients with repeated COS and pregnancy outcomes.

LncRNA XIST Relieves Hypoxia-Induced Damage in H9C2 Cells by Downregulating miR-429

This paper aimed to investigate LncRNA XIST relieving hypoxia-induced damage in H9C2 cells by downregulating miR-429. Rat H9C2 cell lines were selected and divided into a normal control group, a hypoxia group, a XIST expression group, a XIST blank expression group, a miR-429 interference group and a blank interference group. qPCR was adopted for detecting LncRNA XIST and miR-429 expression. Western blot (WB) was adopted for detecting the expression of AMPK, PDH, FAT, MCPT-1, Caspase-3, Bax and Bcl-2, ATP content, and levels of SOD, MDA and LDH. Dual luciferase reporter gene assay (DLRGA) and RNA pull-down were adopted for verifying the correlation of LncRNA XIST with miR-429. Hypoxia-induced H9C2 cells had low LncRNA XIST expression and high miR-429 expression. LncRNA XIST upregulation or miR-429 downregulation could inhibit AMPK, PDH, Caspase-3 and Bax, upregulate FAT, MCPT-1 and Bcl-2, and increase ATP content and SOD activity, as well as reduce MDA content and LDH activity. miR-429 was the target gene of LncRNA XIST. LncRNA XIST can relieve hypoxia-induced damage in H9C2 cells via binding to and downregulating miR-429

Deltamethrin Induces Apoptosis in Cerebrum Neurons of Quail via Promoting Endoplasmic Reticulum Stress and Mitochondrial Dysfunction

Deltamethrin (DLM) is a widely used and highly effective insecticide. DLM exposure is harmful to animal and human. Quail, as a bird model, has been widely used in the toxicology field. However, there is little information available in the literature about quail cerebrum damage caused by DLM. Here, we investigated the effect of DLM on quail cerebrum neurons. Four groups of healthy quails were assigned (10 quails in each group), respectively given 0, 15, 30, and 45 mg/kg DLM by gavage for 12 weeks. Through the measurements of quail cerebrum, it was found that DLM exposure induced obvious histological changes, oxidative stress, and neurons apoptosis. To further explore the possible molecular mechanisms, we performed real-time quantitative PCR to detect the expression of endoplasmic reticulum (ER) stress-related mRNA. In addition, we detected ATP content in quail cerebrum to evaluate the functional status of mitochondria. The study showed that DLM exposure significantly increased the expression of ER stress-related mRNA and decreased ATP content in quail cerebrum. These results suggest that chronic exposure to DLM induces apoptosis of quail cerebrum neurons via promoting ER stress and mitochondrial dysfunction. Furthermore, our results provide a novel explanation for DLM-induced apoptosis of avian cerebrum neurons.