Knockdown of NON-YELLOW COLORING 1 (NYC1)-like gene or chlorophyllin application enhanced chlorophyll accumulation with antioxidant roles in suppressing heat-induced leaf senescence in perennial ryegrass

2021 ◽  
Author(s):  
Guohui Yu ◽  
Zheni Xie ◽  
Wei Chen ◽  
Bin Xu ◽  
Bingru Huang
Keyword(s):  
Leaf Senescence ◽  
Perennial Ryegrass ◽  
Ex Vivo ◽  
Catabolic Gene ◽  
Wild Type ◽  
Mesophyll Protoplasts ◽  
Oxidative Damages ◽  
Catabolic Genes ◽  
Chlorophyll Accumulation ◽  
Sodium Copper Chlorophyllin

Abstract Loss of chlorophyll (Chl) and oxidative damages co-occur during heat-induced leaf senescence. This study aimed to determine the functions of Chl catabolic gene, NON-YELLOW COLORING 1 (NYC1)-like (NOL) in regulating heat-induced leaf senescence and to characterize antioxidant roles of a Chl derivative, sodium copper chlorophyllin (SCC), in suppressing heat-induced leaf senescence. In two separate experiments, one by comparing NOL RNAi transgenic and wild-type plants, and the other by analyzing the effects of sodium copper chlorophyllin (SCC, 1 mM) treatment, perennial ryegrass (Lolium perenne) were exposed to heat stress (38/35 oC, day/night) or optimal temperature (25/20 oC). Results showed that both knockdown of LpNOL and application of SCC suppressed heat-induced leaf senescence, as manifested by increased Chl content, reduced electrolyte leakage, and down-regulation of Chl-catabolic genes and senescence-related genes, as well as enhanced antioxidant capacity in the peroxidase (POD) pathway for H2O2 scavenging. Ex vivo SCC incubation protected membranes from H2O2 damage onto mesophyll protoplasts of perennial ryegrass. The suppression of leaf senescence by knockdown of NOL or chlorophyllin application was associated with enhanced chlorophyll accumulation playing antioxidant roles in protecting leaves from heat-induced oxidative damages.

scholarly journals Melatonin Pretreatment Confers Heat Tolerance and Repression of Heat-Induced Senescence in Tomato Through the Modulation of ABA- and GA-Mediated Pathways

2021 ◽  
Vol 12 ◽  
Author(s):  
Mohammad Shah Jahan ◽  
Sheng Shu ◽  
Yu Wang ◽  
Md. Mahadi Hasan ◽  
Ahmed Abou El-Yazied ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
High Temperature ◽  
Leaf Senescence ◽  
Heat Tolerance ◽  
Catabolic Gene ◽  
Biosynthesis Inhibitor ◽  
Catabolic Genes ◽  
Aba Biosynthesis ◽  
Ga Biosynthesis

Heat stress and abscisic acid (ABA) induce leaf senescence, whereas melatonin (MT) and gibberellins (GA) play critical roles in inhibiting leaf senescence. Recent research findings confirm that plant tolerance to diverse stresses is closely associated with foliage lifespan. However, the molecular mechanism underlying the signaling interaction of MT with GA and ABA regarding heat-induced leaf senescence largely remains undetermined. Herein, we investigated putative functions of melatonin in suppressing heat-induced leaf senescence in tomato and how ABA and GA coordinate with each other in the presence of MT. Tomato seedlings were pretreated with 100 μM MT or water and exposed to high temperature (38/28°C) for 5 days (d). Heat stress significantly accelerated senescence, damage to the photosystem and upregulation of reactive oxygen species (ROS), generating RBOH gene expression. Melatonin treatment markedly attenuated heat-induced leaf senescence, as reflected by reduced leaf yellowing, an increased Fv/Fm ratio, and reduced ROS production. The Rbohs gene, chlorophyll catabolic genes, and senescence-associated gene expression levels were significantly suppressed by MT addition. Exogenous application of MT elevated the endogenous MT and GA contents but reduced the ABA content in high-temperature-exposed plants. However, the GA and ABA contents were inhibited by paclobutrazol (PCB, a GA biosynthesis inhibitor) and sodium tungstate (ST, an ABA biosynthesis inhibitor) treatment. MT-induced heat tolerance was compromised in both inhibitor-treated plants. The transcript abundance of ABA biosynthesis and signaling genes was repressed; however, the biosynthesis genes MT and GA were upregulated in MT-treated plants. Moreover, GA signaling suppressor and catabolic gene expression was inhibited, while ABA catabolic gene expression was upregulated by MT application. Taken together, MT-mediated suppression of heat-induced leaf senescence has collaborated with the activation of MT and GA biosynthesis and inhibition of ABA biosynthesis pathways in tomato.

scholarly journals NTPDase1 Modulates Smooth Muscle Contraction in Mice Bladder by Regulating Nucleotide Receptor Activation Distinctly in Male and Female

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 147
Author(s):  
Romuald Brice Babou Kammoe ◽  
Gilles Kauffenstein ◽  
Julie Pelletier ◽  
Bernard Robaye ◽  
Jean Sévigny
Keyword(s):  
Smooth Muscle ◽  
Ex Vivo ◽  
Smooth Muscle Contraction ◽  
Receptor Activation ◽  
Nucleoside Triphosphate ◽  
Nucleotide Receptors ◽  
Nerve Terminals ◽  
Wild Type ◽  
Nucleoside Triphosphate Diphosphohydrolase ◽  
Bladder Contraction

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5′-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y1, and P2Y6 receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1−/−) mice compared to wild type controls. The strongest responses were obtained with uridine 5′-triphosphate (UTP) and uridine 5′-diphosphate (UDP), suggesting the involvement of P2Y6 receptors, which was confirmed with P2ry6−/− bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y2 and/or P2Y4, and P2Y12 in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1−/−, and P2ry6−/− female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y6 receptor activation were weaker in female bladders.

scholarly journals Cooperative Blockade of CK2 and ATM Kinases Drives Apoptosis in VHL-Deficient Renal Carcinoma Cells through ROS Overproduction

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 576
Author(s):  
Sofia Giacosa ◽  
Catherine Pillet ◽  
Irinka Séraudie ◽  
Laurent Guyon ◽  
Yann Wallez ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Renal Carcinoma ◽  
Kinase Inhibitors ◽  
Ex Vivo ◽  
Cancer Therapeutics ◽  
Carcinoma Cells ◽  
Wild Type ◽  
Cell Renal Cell Carcinoma ◽  
Von Hippel Lindau ◽  
Renal Carcinoma Cells

Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau). A multiplexed assay was used to study the cellular fitness of a panel of engineered ccRCC isogenic 786-O VHL− cell lines in response to a collection of targeted cancer therapeutics including kinase inhibitors, allowing the interrogation of over 2880 drug–gene pairs. Among diverse patterns of drug sensitivities, investigation of the mechanistic effect of one selected drug combination on tumor spheroids and ex vivo renal tumor slice cultures showed that VHL-defective ccRCC cells were more vulnerable to the combined inhibition of the CK2 and ATM kinases than wild-type VHL cells. Importantly, we found that HIF-2α acts as a key mediator that potentiates the response to combined CK2/ATM inhibition by triggering ROS-dependent apoptosis. Importantly, our findings reveal a selective killing of VHL-deficient renal carcinoma cells and provide a rationale for a mechanism-based use of combined CK2/ATM inhibitors for improved patient care in metastatic VHL-ccRCC.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

scholarly journals Pharmacological ablation of astrocytes reduces Aβ degradation and synaptic connectivity in an ex vivo model of Alzheimer’s disease

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Nicola Davis ◽  
Bibiana C. Mota ◽  
Larissa Stead ◽  
Emily O. C. Palmer ◽  
Laura Lombardero ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Culture Media ◽  
Ex Vivo ◽  
Wild Type ◽  
Insulin Degrading Enzyme ◽  
Ex Vivo Model ◽  
Model Of Alzheimer’S Disease ◽  
Aβ Clearance ◽  
5Xfad Mice

Abstract Background Astrocytes provide a vital support to neurons in normal and pathological conditions. In Alzheimer’s disease (AD) brains, reactive astrocytes have been found surrounding amyloid plaques, forming an astrocytic scar. However, their role and potential mechanisms whereby they affect neuroinflammation, amyloid pathology, and synaptic density in AD remain unclear. Methods To explore the role of astrocytes on Aβ pathology and neuroinflammatory markers, we pharmacologically ablated them in organotypic brain culture slices (OBCSs) from 5XFAD mouse model of AD and wild-type (WT) littermates with the selective astrocytic toxin L-alpha-aminoadipate (L-AAA). To examine the effects on synaptic circuitry, we measured dendritic spine number and size in OBCSs from Thy-1-GFP transgenic mice incubated with synthetic Aβ42 or double transgenics Thy-1-GFP/5XFAD mice treated with LAAA or vehicle for 24 h. Results Treatment of OBCSs with L-AAA resulted in an increased expression of pro-inflammatory cytokine IL-6 in conditioned media of WTs and 5XFAD slices, associated with changes in microglia morphology but not in density. The profile of inflammatory markers following astrocytic loss was different in WT and transgenic cultures, showing reductions in inflammatory mediators produced in astrocytes only in WT sections. In addition, pharmacological ablation of astrocytes led to an increase in Aβ levels in homogenates of OBCS from 5XFAD mice compared with vehicle controls, with reduced enzymatic degradation of Aβ due to lower neprilysin and insulin-degrading enzyme (IDE) expression. Furthermore, OBSCs from wild-type mice treated with L-AAA and synthetic amyloid presented 56% higher levels of Aβ in culture media compared to sections treated with Aβ alone, concomitant with reduced expression of IDE in culture medium, suggesting that astrocytes contribute to Aβ clearance and degradation. Quantification of hippocampal dendritic spines revealed a reduction in their density following L-AAA treatment in all groups analyzed. In addition, pharmacological ablation of astrocytes resulted in a decrease in spine size in 5XFAD OBCSs but not in OBCSs from WT treated with synthetic Aβ compared to vehicle control. Conclusions Astrocytes play a protective role in AD by aiding Aβ clearance and supporting synaptic plasticity.

scholarly journals Differential effect of anesthetics on mucociliary clearance in vivo in mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kyle S. Feldman ◽  
Eunwon Kim ◽  
Michael J. Czachowski ◽  
Yijen Wu ◽  
Cecilia W. Lo ◽  
...  
Keyword(s):  
Mucociliary Clearance ◽  
Defense Mechanism ◽  
Ex Vivo ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Wild Type ◽  
Sulfur Colloid ◽  
Ct System

AbstractRespiratory mucociliary clearance (MCC) is a key defense mechanism that functions to entrap and transport inhaled pollutants, particulates, and pathogens away from the lungs. Previous work has identified a number of anesthetics to have cilia depressive effects in vitro. Wild-type C57BL/6 J mice received intra-tracheal installation of 99mTc-Sulfur colloid, and were imaged using a dual-modality SPECT/CT system at 0 and 6 h to measure baseline MCC (n = 8). Mice were challenged for one hour with inhalational 1.5% isoflurane, or intraperitoneal ketamine (100 mg/kg)/xylazine (20 mg/kg), ketamine (0.5 mg/kg)/dexmedetomidine (50 mg/kg), fentanyl (0.2 mg/kg)/1.5% isoflurane, propofol (120 mg/Kg), or fentanyl/midazolam/dexmedetomidine (0.025 mg/kg/2.5 mg/kg/0.25 mg/kg) prior to MCC assessment. The baseline MCC was 6.4%, and was significantly reduced to 3.7% (p = 0.04) and 3.0% (p = 0.01) by ketamine/xylazine and ketamine/dexmedetomidine challenge respectively. Importantly, combinations of drugs containing fentanyl, and propofol in isolation did not significantly depress MCC. Although no change in cilia length or percent ciliation was expected, we tried to correlate ex-vivo tracheal cilia ciliary beat frequency and cilia-generated flow velocities with MCC and found no correlation. Our results indicate that anesthetics containing ketamine (ketamine/xylazine and ketamine/dexmedetomidine) significantly depress MCC, while combinations containing fentanyl (fentanyl/isoflurane, fentanyl/midazolam/dexmedetomidine) and propofol do not. Our method for assessing MCC is reproducible and has utility for studying the effects of other drug combinations.

scholarly journals GPR109A mediates the effects of hippuric acid on regulating osteoclastogenesis and bone resorption in mice

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jin-Ran Chen ◽  
Haijun Zhao ◽  
Umesh D. Wankhade ◽  
Sree V. Chintapalli ◽  
Can Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Mass ◽  
Hippuric Acid ◽  
Ex Vivo ◽  
Marrow Cell ◽  
Osteoclast Differentiation ◽  
Bone Homeostasis ◽  
Wild Type ◽  
Osteoclast Precursors

AbstractThe G protein-coupled receptor 109 A (GPR109A) is robustly expressed in osteoclastic precursor macrophages. Previous studies suggested that GPR109A mediates effects of diet-derived phenolic acids such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA) on promoting bone formation. However, the role of GPR109A in metabolic bone homeostasis and osteoclast differentiation has not been investigated. Using densitometric, bone histologic and molecular signaling analytic methods, we uncovered that bone mass and strength were significantly higher in tibia and spine of standard rodent diet weaned 4-week-old and 6-month-old GPR109A gene deletion (GPR109A−/−) mice, compared to their wild type controls. Osteoclast numbers in bone and in ex vivo bone marrow cell cultures were significantly decreased in GPR109A−/− mice compared to wild type controls. In accordance with these data, CTX-1 in bone marrow plasma and gene expression of bone resorption markers (TNFα, TRAP, Cathepsin K) were significantly decreased in GPR109A−/− mice, while on the other hand, P1NP was increased in serum from both male and female GPR109A−/− mice compared to their respective controls. GPR109A deletion led to suppressed Wnt/β-catenin signaling in osteoclast precursors to inhibit osteoclast differentiation and activity. Indeed, HA and 3-3-PPA substantially inhibited RANKL-induced GPR109A expression and Wnt/β-catenin signaling in osteoclast precursors and osteoclast differentiation. Resultantly, HA significantly inhibited bone resorption and increased bone mass in wild type mice, but had no additional effects on bone in GPR109A−/− mice compared with their respective untreated control mice. These results suggest an important role for GPR109A during osteoclast differentiation and bone resorption mediating effects of HA and 3-3-PPA on inhibiting bone resorption during skeletal development.

scholarly journals KCNJ11 knockout morula re-engineered by stem cell diploid aggregation

2008 ◽  
Vol 364 (1514) ◽  
pp. 269-276 ◽  
Author(s):  
Timothy J Nelson ◽  
Almudena Martinez-Fernandez ◽  
Andre Terzic
Keyword(s):  
Metabolic Flux ◽  
Ex Vivo ◽  
Gene Knockout ◽  
Embryonic Stem ◽  
Adaptive Behaviour ◽  
Wild Type ◽  
New Paradigm ◽  
Cell Functions ◽  
Dependent Cell ◽  
Channel Dependent

KCNJ11 -encoded Kir6.2 assembles with ATP-binding cassette sulphonylurea receptors to generate ATP-sensitive K + (K ATP ) channel complexes. Expressed in tissues with dynamic metabolic flux, these evolutionarily conserved yet structurally and functionally unique heteromultimers serve as high-fidelity rheostats that adjust membrane potential-dependent cell functions to match energetic demand. Genetic defects in channel subunits disrupt the cellular homeostatic response to environmental stress, compromising organ tolerance in the adult. As maladaptation characterizes malignant K ATP channelopathies, establishment of platforms to examine progression of K ATP channel-dependent adaptive behaviour is warranted. Chimeras provide a powerful tool to assay the contribution of genetic variance to stress intolerance during prenatal or post-natal development. Here, KCNJ11 K ATP channel gene knockout↔wild-type chimeras were engineered through diploid aggregation. Integration of wild-type embryonic stem cells into zona pellucida-denuded morula derived from knockout embryos achieved varying degrees of incorporation of stress-tolerant tissue within the K ATP channel-deficient background. Despite the stress-vulnerable phenotype of the knockout, ex vivo derived mosaic blastocysts tolerated intrauterine transfer and implantation, followed by full-term embryonic development in pseudopregnant surrogates to produce live chimeric offspring. The development of adult chimerism from the knockout↔wild-type mosaic embryo offers thereby a new paradigm to probe the ecogenetic control of the K ATP channel-dependent stress response.

scholarly journals N-Acetylcysteine Reduces the Pro-Oxidant and Inflammatory Responses during Pancreatitis and Pancreas Tumorigenesis

Antioxidants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1107
Author(s):  
Marie-Albane Minati ◽  
Maxime Libert ◽  
Hajar Dahou ◽  
Patrick Jacquemin ◽  
Mohamad Assi
Keyword(s):  
Nuclear Translocation ◽  
Inflammatory Responses ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Glutathione Metabolism ◽  
Protective Effects ◽  
Wild Type ◽  
Kras Mutations ◽  
Oxidative Damages ◽  
Factor Α

Pancreatitis, an inflammation of the pancreas, appears to be a main driver of pancreatic cancer when combined with Kras mutations. In this context, the exact redox mechanisms are not clearly elucidated. Herein, we treated mice expressing a KrasG12D mutation in pancreatic acinar cells with cerulein to induce acute pancreatitis. In the presence of KrasG12D, pancreatitis triggered significantly greater redox unbalance and oxidative damages compared to control mice expressing wild-type Kras alleles. Further analyses identified the disruption in glutathione metabolism as the main redox event occurring during pancreatitis. Compared to the wild-type background, KrasG12D-bearing mice showed a greater responsiveness to treatment with a thiol-containing compound, N-acetylcysteine (NAC). Notably, NAC treatment increased the pancreatic glutathione pool, reduced systemic markers related to pancreatic and liver damages, limited the extent of pancreatic edema and fibrosis as well as reduced systemic and pancreatic oxidative damages. The protective effects of NAC were, at least, partly due to a decrease in the production of tumor necrosis factor-α (TNF-α) by acinar cells, which was concomitant with the inhibition of NF-κB(p65) nuclear translocation. Our data provide a rationale to use thiol-containing compounds as an adjuvant therapy to alleviate the severity of inflammation during pancreatitis and pancreatic tumorigenesis.

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