Enzyme Leakage Research Articles

CYTOLOGICAL STUDY ON RAT ISOLATED HEPATOCYTES AND POSSIBLE PROTECTION OF GINGER (EXTRACT AND NANOPARTICLES) AGAINST ACETAMINOPHEN TOXICITY

Ginger is one of most important medicinal plants which used in variable diseases. Recently, ginger nanoparticles have been isolated from ginger extract and exhibited anti-apoptotic activity, hepatoprotective effect against alcohol and may effectively reduce colitis. Our study aimed to compare between the effect of ginger extract and ginger nanoparticles at concentration of 60 µg/ml against hepatotoxicity caused by acetaminophen (APAP; N-acetyl-para-aminophenol) at concentration of 0.1 mg/ml using primary isolated rat hepatocytes. Cytotoxicity was determined by assessing cell viability and leakage of cytosolic enzymes, such as (ALT& AST). Oxidative stress was investigated by measuring levels of MDA and catalase enzymes. The cytopathological lesions were detected by light microscope. Exposure of isolated rat hepatocytes to APAP caused cytotoxicity and oxidative injury, manifested by loss of cell viability and significant increase of enzymes leakages. Also, APAP caused progressive depletion of catalase content and increase in intracellular MDA accumulation, in addition to alteration in histological structures of the cells. Pretreatment of hepatocytes with either GE or GNPs ameliorated the hepatotoxicity, oxidative stress and enzymatic leakage induced by APAP. However, GNPs were more effective compared to ginger extract pre-treated hepatocytes.

Dietary flaxseed's protective effects on body tissues of mice after oral exposure to xylene.

Xylene is a common pollutant in the environment that enters the body of animals and humans in various ways, but most often through the respiratory tract and adversely affects their overall health. However, xylene effects after oral exposure have not been sufficiently studied. This study aimed to investigate the effects of xylene exposure on the mouse organism and to identify possible beneficial effects of flaxseed on such exposure. Eighty mice were divided into four groups: control group C (basal diet + no xylene exposure), group X (oral exposure by 400 mg/kg/day xylene), group F (10% flaxseed supplementation of basal diet), and group XF (10% dietary flaxseed + oral exposure by xylene). Experimental trial took 14 days. Clinical examination, spectroscopic analysis of tissue aminotransferases, total lactate dehydrogenase (TLDH), and acetylcholinesterase (AchE) activities, electrophoretic analysis of LDH isoenzymes, western blot and immunohistochemical analysis of apoptosis as well as routine histology of the kidneys and jejunum, and transmission electron microscopy of the liver were performed. Marked restlessness in group X and high weight losses in mice of all groups were recorded during the experiment. Xylene promoted apoptosis (caspase-3 expression) without causing marked structural changes in the liver and jejunum, although renal cortex structure was affected adversely. In the brain, liver, and kidney of mice, xylene increased levels of liver transaminases, LDH, and decreased AchE activities, reflecting cell membrane damage. Flaxseed feeding improved animal behaviour, leakage of enzymes and prevented selected tissue toxic damage induced by xylene by protecting cell membrane integrity and fluidity and by suppressing apoptosis. These results point at the protective effect of flaxseed consumption on mice.

Plant Growth-Promoting Rhizobacteria Ameliorates Salinity Stress in Pea (Pisum sativum)

Pea is an important legume vegetable crop which is an essential component of human nutrition. Salinity impedes plant growth and productivity of legume crops by disrupting the ionic and osmotic balance and hormonal regulation. The present study was aimed to examine the potential of plant growth-promoting rhizobacteria (PGPR) strains IG 2 (Acinetobacter bereziniae), IG 10 (Enterobacter ludwigii), and IG 27 (Alcaligenes faecalis) in mitigating salinity stress (75 mM and 150 mM NaCl) in pea. Overall the PGPR inoculation (IG 2, IG 10, and IG 27) improved the growth parameters of pea seedling under salinity stress, but PGPR strain IG 27 had better performance than the other two PGPR strains, i.e., IG 10 and IG 2. Similarly, PGPR inoculation modulated the biochemical parameters such as chlorophyll content, proline content, total soluble sugar, electrolyte leakage, and activities of antioxidant enzymes for alleviation of salt stress. Moreover, PGPR-inoculated plants demonstrated lower levels of electrolyte leakage and H2O2 content under saline conditions in relation to un-inoculated pea seedlings. Additionally, PGPR inoculation of pea plant in field trials with 100 mM NaCl stress exhibited enhanced plant growth and yield. Taken together, our findings suggest the beneficial role of PGPR in alleviating the detrimental effect of salinity on pea seedling growth and yield, further could be used as bio-inoculants to improve crop productivity in saline environment.

Nanocomposite-based dual enzyme system for broad-spectrum scavenging of reactive oxygen species

A broad-spectrum reactive oxygen species (ROS)-scavenging hybrid material (CASCADE) was developed by sequential adsorption of heparin (HEP) and poly(L-lysine) (PLL) polyelectrolytes together with superoxide dismutase (SOD) and horseradish peroxidase (HRP) antioxidant enzymes on layered double hydroxide (LDH) nanoclay support. The synthetic conditions were optimized so that CASCADE possessed remarkable structural (no enzyme leakage) and colloidal (excellent resistance against salt-induced aggregation) stability. The obtained composite was active in decomposition of both superoxide radical anions and hydrogen peroxide in biochemical assays revealing that the strong electrostatic interaction with the functionalized support led to high enzyme loadings, nevertheless, it did not interfere with the native enzyme conformation. In vitro tests demonstrated that ROS generated in human cervical adenocarcinoma cells were successfully consumed by the hybrid material. The cellular uptake was not accompanied with any toxicity effects, which makes the developed CASCADE a promising candidate for treatment of oxidative stress-related diseases.

Therapeutic efficacy of a Dioscorea membranacea extract in a rat model of hepatocellular carcinoma: Histopathological aspects

Hepatocellular carcinoma (HCC) is most common in adults and has a high mortality rate because of a lack of effective treatment options. We investigated the effect of a medicinal plant as a potential source of drugs against HCC. The rhizomes of Dioscorea membranacea Pierre (DM), Hua-Khao-Yen in Thai, are commonly used as ingredients for alternative treatment of cancer in Thailand. In this study, the anticancer effects of DM extract in HCC-bearing rats were evaluated with respect to gross morphology, histopathology, and leakage of liver enzymes. In untreated HCCs, typical features of liver cancer, including hepatic nodules, thick-cell cords, and pseudoglandular cell arrangements, were observed. In addition, the HCCs showed abnormal reticulin patterns and a high glypican3 expression. In HCC-bearing rats treated with DM the cancer areas and reticulin expression were significantly reduced compared to the untreated group (p < 0.01). Sorafenib, the standard drug to treat HCC, reduced the cancer area further, but increased leakage of liver enzymes and decreased serum albumin concentration, indicating liver toxicity. These findings suggest that DM has an anticancer effect on HCCs in an animal model in vivo with potentially less severe side effects than sorafenib. Therefore, further studies of DM’s mechanism of action in HCC should be carried out.

Relation between washed sperm cells and fluid from White Fulani Genitalia in some enzyme activities and lipid contents

Lipid contents and enzyme activities were studied in the fluid and washed sperm (WS) from the genitalia of 26 mature White Fulani (WF) bulls of mean live weight 213.96±4.88kg (range 140 260kg) in a complete randomized design experiment. Tissue homogenization and biochemical assay methods were employed after storing the tissue in deep freeze for 24 hours post slaughter. Total lipid contents did not differ significantly (P&gt;0.05) between WS and fluid of the testis, the caput epididymis and the cauda epididymis but was significantly (P&lt;0.05) higher in WS than the fluid in corpus epididymis and vas deferens. Phospholipid and phospholipid phosphorus contents of WS and fluid did not vary significantly (P&gt;0.05) benveen WS in the testis and epididymis. Alkaline phosphatatse (ALP) activity was higher (P&lt;0.05) in the corpus and cauda edididymal fluid than in the WS. Glutamic Oxalate Transaminase (GOT) activity was significantly (P&lt;0.05) higher in the fluid than the WS in all segments but not in the testis. The generally higher values of lipids in the washed sperm over the fluid the need for lipids as protective cover and as substrates for sperm survival from its formation up to maturation. The generally higher enzyme activity in the fluid is an indicator of damage to sperm acrosome, with corresponding leakage of enzyme into the fluid consequent upon the various stress conditions encountered as sperm traverses the epididymis for the maturation process and finally sojourns in the epididymis.

Role of molecular and metabolic defects in impaired performance of dystrophic skeletal muscles

There occurs a progressive weakness and wastage of skeletal muscle in different types of muscular dystrophy. The loss of muscle fibers in dystrophic muscle with impaired function is associated with leakage of intracellular enzymes, maldistribution of electrolyte content and metabolic defects in myocytes. Marked increases in the sarcolemma (SL) Na+-K+ ATPase and Ca2+/Mg2+-ecto ATPase activities, as well as depressions in the sarcoplasmic reticulum (SR) Ca2+-uptake and Ca2+-pump ATPase activities were seen in dystrophic muscles of a hamster model of myopathy. In addition, impaired mitochondrial oxidative phosphorylation and decrease in the high energy stores as a consequence of mitochondrial Ca2+-overload were observed in these myopathic hamsters. In some forms of muscular dystrophy, it has been shown that deficiency of dystrophin produces marked alterations in the SL permeability and promotes the occurrence of intracellular Ca2+-overload for inducing metabolic defects, activation of proteases and contractile abnormalities in dystrophic muscle. Increases in SR Ca2+-release channels, SL Na+-Ca2+ exchanger and SL store-operated Ca2+-channels have been reported to induce Ca2+-handling abnormalities in a mouse model of muscular dystrophy. Furthermore, alterations in lipid metabolism and development of oxidative stress have been suggested as mechanisms for subcellular remodeling and cellular damage in dystrophic muscle. Although, several therapeutic interventions including gene therapy are available, these treatments neither fully prevent the course of development of muscular disorder nor fully improve the function of dystrophic muscle. Thus, extensive reasearch work with some novel inhibitors of oxidative stress, SL Ca2+-entry systems such as store-operated Ca2+-channels, Na+-Ca2+ exchanger and Ca2+/Mg2+-ecto ATPase (Ca2+-gating mechanism), as well as SR Ca2+-release and Ca2+-pump systems needs to be carried out in combination of gene therapy for improved beneficial effects in muscular dystrophy.

Ameliorating Effect of Essential Phospholipids Enriched with Virgin Coconut oil (Phoscoliv®) on Alcohol Induced Liver Toxicity: Possible Role in Oxidative Stress and Cellular Leakage

Background: Researchers had shown that essential phospholipids and virgin coconut oil are the very effective agents used for the treatment of several liver disorders. Thus, the current study aimed to evaluate the ameliorating effect of Phoscoliv (PL), a novel formulation using the combination of essential phospholipids enriched with virgin coconut oil. Methods: In the study, adult wistar rats were grouped into three as Normal (N), Ethanol Treated (ET) (12.5 g/ kg body weight of 90% [v/v]) and ET+ PL (0.5 mL/100g body weight) for 30 days. Chronic administration of ethanol induced severe damage to the liver which leads to the imbalance of normal cellular and metabolic activities. It was determined by evaluating the antioxidant status, liver marker enzymes, lipid peroxidation product, pro-inflammatory cytokines and histopathological analysis. Results: Results revealed that the supplementation of PL enhance the antioxidants thereby reduced the oxidative stress associated liver damages by inhibiting lipid peroxidation product, pro-inflammatory cytokines release and leakage of liver marker enzymes. Thereby, improve the regeneration capacity of hepatic cells and maintain its normal functioning. Conclusion: Hence, PL exhibited its potent hepatoprotective activity as well as antioxidant effect against alcohol induced liver toxicity.

When lipid homeostasis runs havoc: Lipotoxicity links lysosomal dysfunction to autophagy

Autophagy is one of the major cellular degradation pathways, which prevents accumulation of cellular wastes including "hazardous" material such as oxidized proteins and lipids and allows removal of aggregates and dysfunctional organelles. Hence, autophagy is meant to preserve cell survival, and is mostly protective. However, autophagy may trigger a feedforward, exaggerated cycle in which cells continue to degrade proteins and organelles, finally leading to autophagy-dependent cell death (ADCD), a process that can be initiated with lysosomotropic detergents, which are protonated within the lysosome and cause a permeabilization of the membrane. Such drugs may be useful to combat cancer. In some paradigms of ADCD, there is evidence that the cellular fate is determined by the integrity of lysosomal membranes, transporters, enzymes and ion gradients. Detergent-like effects of lysosomotropic drugs can over-activate autophagy. A disruption of the lysosomal membrane barrier with leakage of lysosomal enzymes or lipids may trigger a vicious cycle via proteases and accumulation of lipids, which impair the functions of the plasma - and organelle membranes. This review summarizes the current evidence for a crosstalk between lysosomal dysfunction and autophagy and the lysosomal events, which progress toward ADCD with a focus on the role of sphingolipids and cholesterol as cargo and as regulators of ADCD.

Confining the motion of enzymes in nanofiltration membrane for efficient and stable removal of micropollutants

Enzymes in living cells are highly dynamic but at the same time regularly confined for achieving efficient metabolism. Inspired by this phenomenon, we have prepared a novel biocatalytic membrane with high enzyme activity and stability by tuning the confinement strength of the membrane to enzyme, which was achieved via modifying the support layer of a polymeric nanofiltration (NF) membrane and reversely filtrating enzyme. A mussel-inspired coating was used to modify the support interior of the NF membrane to enhance charge and steric effects on enzyme, thus stabilizing enzyme in the membrane with little increment in mass transfer resistance for substrate and products (only 20% permeability loss with a high enzyme loading of 1.34 mg/cm2). A suitable confinement strength of the membrane to enzyme could delay the enzyme leakage and endow enzyme with certain mobility for efficient reaction. Thus, the obtained biocatalytic membrane exhibited a negligible decline in BPA removal efficiency for 7 reuse cycles (<3.5%) or 36 h continuous operation (<1%) in flow through mode, resulting in a long-term stability adequate for micropollutant removal. For the first time, enzyme mobility was defined and calculated to quantify the confinement strength of the membrane, which could be used to optimize the microenvironment for enzyme immobilization and predict the performance of the biocatalytic membrane. This work concluded that rationally regulating the enzyme mobility in the membrane and a periodic back-flushing operation for redistribution of enzymes could achieve a long-term stable removal of micropollutant in water by a biocatalytic membrane.

(Invited) Glucose/Oxygen Wearable Biofuel Cells Based on Poly(glycidyl methacrylate)-Grafted MgO-Templated Porous Carbon

There are a lot of recent researches on high power enzymatic glucose/O2 biofuel cells (BFCs). We have reported that the use of MgO-templated porous carbon (MgOC) at the electrodes of biofuel cells dramatically improves the output power [1-4]. Using MgOC can improve the effective electrode surface area, leading to increased current density and output density. Supporting enzymes on these types of porous materials is expected to lead to increased output density. In the past researches [1-4], the enzymes physically adsorbed inside the porous carbon gradually leach from the carbon surface after long-term operation or during the replacement of the fuel solution.In this study, to prevent enzyme leakage, the enzyme or mediator were immobilized on the MgOC surface via multipoint covalent bonding; the grafting of polymers bearing pendant glycidyl groups was achieved by the polymerization of glycidyl methacrylate (GMA), initiated by electron beam irradiation on the carbon surface. Poly(glycidyl methacrylate) (poly(GMA)) bearing pendant glycidyl groups, grafted on the surface of MgO-templated carbon, is useful for forming strong multipoint covalent bonds with amino functional groups on the surface of flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH), Azure A and aminoferrocene.For example, the immobilized FAD-GDH can generate glucose-oxidation catalytic current using 1,2-naphthoquinone (12NQ) as the redox mediator, which is also captured on the electrode surface. The catalytic current is more stable than that obtained using the FAD-GDH-MgOC electrode without poly(GMA) because the leaching of FAD-GDH and 12NQ is suppressed by the enzyme capping effect.The BFC performance using the anode measured in 1.0 M phosphate buffer (PBS) containing 100 mM glucose exhibited the open circuit voltage of 0.8 V, maximum power density of 1.4 mW/cm2, and maximum current density of 4.25 mA/cm2.In the present paper, we totally discuss three types glucose/oxygen biofuel cells based on the poly(glycidyl methacrylate)-grafted MgO-templated porous carbon.

Biodegradation of cyanide in cassava wastewater using a novel thermodynamically-stable immobilized rhodanese

Extracellular rhodanese obtained from Aureobasidium pullulans was employed in both free and immobilized forms for the biodegradation of cyanide present in cassava processing mill effluent (CPME). Crosslinking with glutaraldehyde (at an optimum concentration of 5% v/v) before entrapment in alginate beads resulted in the highest immobilization yield of 94.5% and reduced enzyme leakage of 1.8%. Rhodanese immobilized by cross-linking before entrapment (cbe) retained about 46% of its initial activity after eight cycles of catalysis compared to the entrapment in alginate alone (eaa) which lost more than 79% after the fifth catalytic cycle. A cross-examination of thermodynamic ( ) kinetic () parameters at 30–70 °C showed that cbe displayed a higher resistance to thermal inactivation when compared to the free enzyme (fe) and (eaa). The efficiency of cyanide biodegradation from the CPME by the fe, eaa and cbe were 55, 62, and 74% respectively after 6 h. Rhodanese immobilized via cbe had a higher resistance to thermal denaturation over other enzyme forms. Hence, this makes cbe adaptable for large-scale detoxification of cyanide from CPME.

Multienzyme Cellulose Films as Sustainable and Self-Degradable Hydrogen Peroxide-Producing Material.

The use of hydrogen peroxide-releasing enzymes as a component to produce alternative and sustainable antimicrobial materials has aroused interest in the scientific community. However, the preparation of such materials requires an effective enzyme binding method that often involves the use of expensive and toxic chemicals. Here, we describe the development of an enzyme-based hydrogen peroxide-producing regenerated cellulose film (RCF) in which a cellobiohydrolase (TrCBHI) and a cellobiose dehydrogenase (MtCDHA) were efficiently adsorbed, 90.38 ± 2.2 and 82.40 ± 5.7%, respectively, without making use of cross-linkers. The enzyme adsorption kinetics and binding isotherm experiments showed high affinity of the proteins possessing cellulose-binding modules for RCF, suggesting that binding on regenerated cellulose via specific interactions can be an alternative method for enzyme immobilization. Resistance to compression and porosity at a micrometer scale were found to be tunable by changing cellulose concentration prior to film regeneration. The self-degradation process, triggered by stacking TrCBHI and MtCDHA (previously immobilized onto separate RCF), produced 0.15 nmol/min·cm2 of H2O2. Moreover, the production of H2O2 was sustained for at least 24 h reaching a concentration of ∼2 mM. The activity of MtCDHA immobilized on RCF was not affected by reuse for at least 3 days (1 cycle/day), suggesting that no significant enzyme leakage occurred in that timeframe. In the material herein designed, cellulose (regenerated from a 1-ethyl-3-methylimidazolium acetate/dimethyl sulfoxide (DMSO) solution) serves both as support and substrate for the immobilized enzymes. The sequential reaction led to the production of H2O2 at a micromolar-millimolar level revealing the potential use of the material as a self-degradable antimicrobial agent.

The Modulatory Role of Vitamin C in Boldenone Undecylenate Induced Testicular Oxidative Damage and Androgen Receptor Dysregulation in Adult Male Rats.

Background: This study explored the effect of vitamin C (Vit-C) administration on the reproductive function of adult male Wistar rats injected with boldenone undecylenate (BOL). Methods: Rats were randomly assigned into control, vehicle control, Vit-C (120 mg/kg b.wt./day, orally), BOL (received 5 mg/kg b.wt./week, IM) and BOL+Vit-C-treated groups. After eight weeks, hormonal assay, semen evaluation, testicular enzymes, and antioxidants biomarkers were assessed. Besides, the histopathological and immunohistochemical investigations of the androgen receptor (AR) expression were performed. Results: The results revealed that serum testosterone, acid phosphatase, sorbitol dehydrogenase, sperm abnormalities, and testicular malondialdehyde were significantly incremented in the BOL-treated group. Testicular weight, sperm count, and sperm motility together with serum levels of luteinizing hormone, follicle-stimulating hormone, and estradiol, and testicular testosterone, catalase, superoxide dismutase, and reduced glutathione showed a significant decrease following BOL treatment. Besides, the AR immunoreactivity was significantly decreased in testicular tissues. Vit-C co-administration with BOL significantly relieved the BOL-induced sperm abnormalities, reduced sperm motility, testicular enzyme leakage, and oxidative damage. However, Vit-C could rescue neither BOL-induced hormonal disturbances nor AR down-regulation. Conclusions: The results provide further insight into the mechanisms of BOL-induced reproductive dysfunction and its partial recovery by Vit-C.

Investigation of the efficacy of mycotoxin-detoxifying additive on health and growth of newly-weaned pigs under deoxynivalenol challenges.

ObjectiveThis study evaluated the effects of feeding diets naturally contaminated with deoxynivalenol (supplemental 2 mg/kg) on health, growth, and the effects of a mycotoxin-detoxifying additive in newly-weaned pigs.MethodsThirty-six pigs (27 day-old) were housed individually and assigned to 3 treatments for 5 weeks: CON (diet containing minimal deoxynivalenol), MT (diet with supplemental 1.9 mg/kg of deoxynivalenol), and MT+D (MT + mycotoxin-detoxifying additive, 0.2%, MegaFix, ICC, São Paulo, Brazil). The mycotoxin-detoxifying additive included bentonite, algae, enzymes, and yeast. Blood was taken at week 2 and 5. Jejunal tissue were taken at week 5. Data were analyzed using the MIXED procedure of SAS.ResultsPigs fed MT+D tended to have decreased (p = 0.056) averaged daily feed intake during week 1 than MT. At week 2, serum aspartate aminotransferase/alanine aminotransferase in MT tended to be lower (p = 0.059) than CON, whereas it was increased (p< 0.05) for MT+D than MT, indicating hepatic damages in MT and recovery in MT+D. Pigs fed MT had lower (p<0.05) blood urea nitrogen/creatinine than CON, supporting hepatic damage. At week 5, pigs fed MT tended to have reduced (p = 0.079) glucose than CON, whereas it was increased (p<0.05) for MT+D than MT, indicating impaired intestinal glucose absorption in MT, which was improved in MT+D. Pigs fed CON tended to have increased (p = 0.057) total glutathione in jejunum than MT, indicating oxidative stress in MT. Pigs fed MT+D had a reduced (p<0.05) proportion of Ki-67-positive cells in jejunum than MT, indicating lower enterocyte proliferation in MT+D.ConclusionFeeding supplemental 1.9 mg/kg of deoxynivalenol reduced growth and debilitated hepatic health of pigs, as seen in leakage of hepatic enzymes, impaired nitrogen metabolism, and increase in oxidative stress. The mycotoxin-detoxifying enhanced hepatic health and glucose levels, and attenuated gut damage in pigs fed deoxynivalenol contaminated diets.

Oxidative Stress and Apoptosis Contributed to Nonylphenol-Induced Cell Damage in Mouse NCTC Clone 1469 Cells

Nonylphenol (NP) is considered an environmental toxicant and endocrine-disrupting compound. The present study aimed to investigate the effects of NP on NCTC Clone 1469, nonparenchymal hepatocytes, and to study the molecular basis of NP-induced liver injury. The results showed that NP decreased cell viability and induced nucleus crenulation and intracellular enzyme leakage in NCTC Clone 1469 cells. Additionally, NP-induced oxidative stress and apoptosis of NCTC Clone 1469 are accompanied by upregulating reactive oxygen species (ROS) production, increase of Bax, decrease of Bcl-2, activation of caspase-3 and caspase-12, and release of cytosolic free Ca2+ in the cells. ROS scavenger, N-acetyl-L-cysteine (NAC), prevented the intracellular enzyme leakage induced by NP. NP induced alteration of estrogen receptor- (ER-) α and ER-β expression, while ER antagonists, ICI 182,780, showed no effect on NP-induced intracellular enzyme leakage. We proposed that NP triggered cell damage via inducing oxidative stress and apoptosis in cells, but not estrogenic effect.

Triacontanol Protects Mentha arvensis L. from Nickel-Instigated Repercussions by Escalating Antioxidant Machinery, Photosynthetic Efficiency and Maintaining Leaf Ultrastructure and Root Morphology

Nickel (Ni), an essential micronutrient and a prime component of the plant enzyme urease, has an indispensable role in plants. Triacontanol (TRIA) is a conspicuous plant growth regulator in agriculture, which proved advantageous in enhancing the overall production of plants. Therefore, an experiment was laid down to understand the effects of Ni toxicity on the menthol mint (Mentha arvensis L.) and its mitigation by exogenously applied TRIA. The different treatments applied to the plants were 0 (control), TRIA (10−6 M), Ni (60 mg kg−1), Ni (80 mg kg−1), TRIA (10−6 M) + Ni (60 mg kg−1), and TRIA (10−6 M) + Ni (80 mg kg−1). This work was evaluated on the basis of various growth, biochemical, physiological, yield and quality parameters. Nickel applied at 80 mg kg−1 of soil exhibited maximum inhibition in the parameters studied. Application of TRIA improved all the growth parameters such as plant height, fresh and dry weights as well as herbage yield under non stress and stressed conditions. The levels of carbonic anhydrase (CA) activity, photosynthetic parameters (chlorophyll and carotenoids), and chlorophyll fluorescence of the plants were also stimulated by TRIA under Ni stress. Exogenous TRIA also displayed positive effects on the cellular antioxidant defense mechanism of Ni-affected plants as it increased the levels of proline (PRO), electrolytic leakage (EL), and activities of antioxidant enzymes, viz. superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX), therefore, restrained the triggering of the oxidative burst (reactive oxygen species) in the plant cells. Moreover, TRIA improved the overall production (in terms of yield and content) of EO in the plants and maintained the leaf ultrastructure and root morphology under Ni treatment. GC–MS analysis revealed that TRIA upregulated the level of menthone and menthyl acetate over their respective controls and under Ni-stressed condition.

Integrated transcriptomic and metabolomic analyses of yellow horn (Xanthoceras sorbifolia) in response to cold stress.

Xanthoceras sorbifolia, a medicinal and oil-rich woody plant, has great potential for biodiesel production. However, little study explores the link between gene expression level and metabolite accumulation of X. sorbifolia in response to cold stress. Herein, we performed both transcriptomic and metabolomic analyses of X. sorbifolia seedlings to investigate the regulatory mechanism of resistance to low temperature (4 °C) based on physiological profile analyses. Cold stress resulted in a significant increase in the malondialdehyde content, electrolyte leakage and activity of antioxidant enzymes. A total of 1,527 common differentially expressed genes (DEGs) were identified, of which 895 were upregulated and 632 were downregulated. Annotation of DEGs revealed that amino acid metabolism, glycolysis/gluconeogenesis, starch and sucrose metabolism, galactose metabolism, fructose and mannose metabolism, and the citrate cycle (TCA) were strongly affected by cold stress. In addition, DEGs within the plant mitogen-activated protein kinase (MAPK) signaling pathway and TF families of ERF, WRKY, NAC, MYB, and bHLH were transcriptionally activated. Through metabolomic analysis, we found 51 significantly changed metabolites, particularly with the analysis of primary metabolites, such as sugars, amino acids, and organic acids. Moreover, there is an overlap between transcript and metabolite profiles. Association analysis between key genes and altered metabolites indicated that amino acid metabolism and sugar metabolism were enhanced. A large number of specific cold-responsive genes and metabolites highlight a comprehensive regulatory mechanism, which will contribute to a deeper understanding of the highly complex regulatory program under cold stress in X. sorbifolia.

Hyperpolarized [1,4-13C]fumarate imaging detects microvascular complications and hypoxia mediated cell death in diabetic nephropathy

Today, there is a general lack of prognostic biomarkers for development of renal disease and in particular diabetic nephropathy. Increased glycolytic activity, lactate accumulation and altered mitochondrial oxygen utilization are hallmarks of diabetic kidney disease. Fumarate hydratase activity has been linked to mitochondrial dysfunction as well as activation of the hypoxia inducible factor, induction of apoptosis and necrosis. Here, we investigate fumarate hydratase activity in biofluids in combination with the molecular imaging probe, hyperpolarized [1,4-13C2]fumarate, to identify the early changes associated with hemodynamics and cell death in a streptozotocin rat model of type 1 diabetes. We found a significantly altered hemodynamic signature of [1,4-13C2]fumarate in the diabetic kidneys as well as an systemic increased metabolic conversion of fumarate-to-malate, indicative of increased cell death associated with progression of diabetes, while little to no renal specific conversion was observed. This suggest apoptosis as the main cause of cell death in the diabetic kidney. This is likely resulting from an increased reactive oxygen species production following uncoupling of the electron transport chain at complex II. The mechanism coupling the enzyme leakage and apoptotic phenotype is hypoxia inducible factor independent and seemingly functions as a protective mechanism in the kidney cells.

Stability-Controllable Self-Immobilization of Carbonic Anhydrase Fused with a Silica-Binding Tag onto Diatom Biosilica for Enzymatic CO2 Capture and Utilization.

Exploiting carbonic anhydrase (CA), an enzyme that catalyzes the hydration of CO2, is a powerful route for eco-friendly and cost-effective carbon capture and utilization. For successful industrial applications, the stability and reusability of CA should be improved, which necessitates enzyme immobilization. Herein, the ribosomal protein L2 (Si-tag) from Escherichia coli was utilized for the immobilization of CA onto diatom biosilica, a promising renewable support material. The Si-tag was redesigned (L2NC) and genetically fused to CA from the marine bacterium Hydrogenovibrio marinus (hmCA). One-step self-immobilization of hmCA-L2NC onto diatom biosilica by simple mixing was successfully achieved via Si-tag-mediated strong binding, showing multilayer adsorption with a maximal loading of 1.4 wt %. The immobilized enzyme showed high reusability and no enzyme leakage even under high temperature conditions. The activity of hmCA-L2NC was inversely proportional to the enzyme loading, while the stability was directly proportional to the enzyme loading. This discovered activity-stability trade-off phenomenon could be attributed to macromolecular crowding on the highly dense surface of the enzyme-immobilized biosilica. Collectively, our system not only facilitates the stability-controllable self-immobilization of enzyme via Si-tag on a diatom biosilica support for the robust, facile, and green construction of stable biocatalysts, but is also a unique model for studying the macromolecular crowding effect on surface-immobilized enzymes.