Non-enzymatic Mechanisms Research Articles

P0062GLUCOSYLCERAMIDE SYNTHASE INHIBITION WITH VENGLUSTAT IN CLASSIC FABRY DISEASE PATIENTS LEADS TO PROGRESSIVE REDUCTION OF ENDOTHELIAL CELL GLOBOTRIAOSYLCERAMIDE INCLUSION VOLUME

Abstract Background and Aims Fabry disease (FD) is a rare, X-linked, genetic disorder caused by mutations in the gene for the lysosomal enzyme alpha-galactosidase A (αGal-A). The progressive accumulation of glycosphingolipids, most notably globotriaosylceramide (GL-3), in lysosomes of a variety of organs and cell types leads, over decades, to renal, cardiovascular, and other clinical manifestations. In a phase 2 study, glucosylceramide synthase inhibition with venglustat led to a progressive reduction in plasma GL-3 to low-normal levels. Scoring by light microscopy showed a reduction in lysosomal GL-3 inclusions in skin capillary endothelial cells (EC) after 3 years of therapy, although not after 6 months. To further characterize the effect of venglustat using more precise methods, we applied unbiased stereological methods to quantitate GL-3 inclusions in skin ECs. Methods Skin biopsies were obtained from classic male Fabry disease patients (N = 11) at baseline and during daily treatment with venglustat in a phase 2 study (NCT02228460, NCT02489344). Images from at least 50 superficial skin capillaries were obtained using transmission electron microscopy at 7,500 X magnification. The fraction of the volume (Vv) of cytoplasm occupied by GL-3 inclusions [Vv(Inc/Endo)] in all ECs in these capillaries was estimated using point counting by a masked reader. Two-sided paired t tests were used to evaluate differences between baseline and post-treatment values at each time point. Results Venglustat therapy led to a significant reduction from baseline in Vv(Inc/Endo) of 21.1% after 6 months and 38.7% after 3 years of treatment (Figure). Conclusions Treatment with venglustat led to a reduction in GL-3 inclusion fractional volume in skin capillary ECs which was detectable after 6 months using precise quantitative methods. This was followed by further reduction over the next 2 1/2 years. We posit that, in the absence of αGal-A activity, inhibition of GL-3 production with venglustat allowed other enzymatic or non-enzymatic mechanisms to progressively reduce EC lysosomal GL-3 content, and that long-term venglustat therapy may therefore prevent or reverse progressive tissue injury in Fabry disease.

Computational studies on nonenzymatic succinimide-formation mechanisms of the aspartic acid residues catalyzed by two water molecules

In the biological proteins, aspartic acid (Asp) residues are prone to nonenzymatic isomerization via a succinimide (Suc) intermediate. Asp-residue isomerization causes the aggregation and the insolubilization of proteins, and is considered to be involved in various age-related diseases. Although Suc intermediate was considered to be formed by nucleophilic attack of the main-chain amide nitrogen of N-terminal side adjacent residue to the side-chain carboxyl carbon of Asp residue, previous studies have shown that the nucleophilic attack is more likely to proceed via iminol tautomer when the water molecules act as catalysts. However, the full pathway to Suc-intermediate formation has not been investigated, and the experimental analyses for the Asp-residue isomerization mechanism at atomic and molecular levels, such as the analysis of the transition state geometry, are difficult. In the present study, we computationally explored the full pathways for Suc-intermediate formation from Asp residues. The calculations were performed two types of reactant complexes, and all energy minima and TS geometries were optimized using B3LYP density functional methods. As a result, the SI-intermediate formation was divided into three processes, i.e., iminolization, cyclization, and dehydration processes, and the activation energies were calculated to be 26.1 or 28.4 kcal mol−1. These values reproduce the experimental data. The computational results show that abundant water molecules in living organisms are effective catalysts for the Asp-residue isomerization.

Ectomycorrhizal Fungi: Participation in Nutrient Turnover and Community Assembly Pattern in Forest Ecosystems

Ectomycorrhizal fungi (EcMF) are involved in soil nutrient cycling in forest ecosystems. These fungi can promote the uptake of nutrients (e.g., nitrogen (N) and phosphorus (P)) and water by host plants, as well as facilitate host plant growth and resistance to stresses and diseases, thereby maintaining the aboveground primary productivity of forest ecosystems. Moreover, EcMF can acquire the carbon (C) sources needed for their growth from the host plants. The nutrient regulation mechanisms of EcMF mainly include the decay of soil organic matter via enzymatic degradation, nonenzymatic mechanism (Fenton chemistry), and priming effects, which in turn promote C and N cycling. At the same time, EcMF can secrete organic acids and phosphatases to improve the availability of soil P, or increase mycelium inputs to facilitate plant acquisition of P. The spatiotemporal distribution of EcMF is influenced by a combination of historical factors and contemporary environmental factors. The community of EcMF is associated with various factors, such as climate change, soil conditions, and host distribution. Under global climate change, investigating the relationships between the nutrient cycling functions of EcMF communities and their distribution patterns under various spatiotemporal scales is conducive to more accurate assessments of the ecological effects of EcMF on the sustainable development of forest.

Oxidized Phospholipids in Healthy and Diseased Lung Endothelium.

Circulating and cell membrane phospholipids undergo oxidation caused by enzymatic and non-enzymatic mechanisms. As a result, a diverse group of bioactive oxidized phospholipids generated in these conditions have both beneficial and harmful effects on the human body. Increased production of oxidized phospholipid products with deleterious effects is linked to the pathogenesis of various cardiopulmonary disorders such as atherosclerosis, thrombosis, acute lung injury (ALI), and inflammation. It has been determined that the contrasting biological effects of lipid oxidation products are governed by their structural variations. For example, full-length products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine oxidation (OxPAPC) have prominent endothelial barrier protective and anti-inflammatory activities while most of the truncated oxidized phospholipids induce vascular leak and exacerbate inflammation. The extensive studies from our group and other groups have demonstrated a strong potential of OxPAPC in mitigating a wide range of agonist-induced lung injuries and inflammation in pulmonary endothelial cell culture and rodent models of ALI. Concurrently, elevated levels of truncated oxidized phospholipids are present in aged mice lungs that potentiate the inflammatory agents-induced lung injury. On the other hand, increased levels of full length OxPAPC products accelerate ALI recovery by facilitating production of anti-inflammatory lipid mediator, lipoxin A4, and other molecules with anti-inflammatory properties. These findings suggest that OxPAPC-assisted lipid program switch may be a promising therapeutic strategy for treatment of acute inflammatory syndromes. In this review, we will summarize the vascular-protective and deleterious aspects of oxidized phospholipids and discuss their therapeutic potential including engineering of stable analogs of oxidized phospholipids with improved anti-inflammatory and barrier-protective properties.

Mechanisms and Treatment of Light-Induced Retinal Degeneration-Associated Inflammation: Insights from Biochemical Profiling of the Aqueous Humor.

Ocular inflammation contributes to the pathogenesis of blind-causing retinal degenerative diseases, such as age-related macular degeneration (AMD) or photic maculopathy. Here, we report on inflammatory mechanisms that are associated with retinal degeneration induced by bright visible light, which were revealed while using a rabbit model. Histologically and electrophysiologically noticeable degeneration of the retina is preceded and accompanied by oxidative stress and inflammation, as evidenced by granulocyte infiltration and edema in this tissue, as well as the upregulation of total protein, pro-inflammatory cytokines, and oxidative stress markers in aqueous humor (AH). Consistently, quantitative lipidomic studies of AH elucidated increase in the concentration of arachidonic (AA) and docosahexaenoic (DHA) acids and lyso-platelet activating factor (lyso-PAF), together with pronounced oxidative and inflammatory alterations in content of lipid mediators oxylipins. These alterations include long-term elevation of prostaglandins, which are synthesized from AA via cyclooxygenase-dependent pathways, as well as a short burst of linoleic acid derivatives that can be produced by both enzymatic and non-enzymatic free radical-dependent mechanisms. The upregulation of all oxylipins is inhibited by the premedication of the eyes while using mitochondria-targeted antioxidant SkQ1, whereas the accumulation of prostaglandins and lyso-PAF can be specifically suppressed by topical treatment with cyclooxygenase inhibitor Nepafenac. Interestingly, the most prominent antioxidant and anti-inflammatory benefits and overall retinal protective effects are achieved by simultaneous administrating of both drugs indicating their synergistic action. Taken together, these findings provide a rationale for using a combination of mitochondria-targeted antioxidant and cyclooxygenase inhibitor for the treatment of inflammatory components of retinal degenerative diseases.

Involvement of Heparanase in Gastric Cancer Progression and Immunotherapy.

Heparanase is upregulated in various tumors, and its expression is closely associated with tumor growth, angiogenesis and metastasis, which accomplishes this mainly through degrading heparan sulfate and releasing heparin-binding growth factors thereby influencing multiple signaling pathways. In addition to its enzymatic degrading activity, heparanase can act via its non-enzymatic mechanisms that directly regulate various signaling. This review mainly focuses on the expression levels and role of heparanase in gastric cancer, and multiple genes and mechanisms regulating heparanase expression in gastric cancer. Furthermore, the development of heparanase-targeted immunotherapy and its potential application for treating gastric cancer are discussed.

Nitric Oxide: The Key Molecule for Polyphenols Antimicrobial Action

The role of hydroxycinnamic acids as antioxidants, in vivo, has been widely discussed, but, recently, a great debate has focused on their antimicrobial action. In general, for the hydroxycinnamic acids’ action, the presence of NO, which is known to be an antimicrobial agent, seems compulsory; its production goes through the intermediacy of the nitrosonium ion, and a very low pH, for instance, as in the stomach, is requested. However, the action of the hydroxycinnamic acids seems to take place even in different biological compartments, i.e., characterized by different pHs and conditions, and then, for NO production, an alternative mechanism could be involved. In this light, evidence for the NO formation, via an E.T. mechanism, even in mildly acidic conditions (pH = 6.4), was obtained by reacting an aqueous buffer solution of acidic nitrite (HNO2) with the hydroxycinnamic acids ferulic, caffeic, p-coumaric and sinapic. Experiments conducted by EPR spectroscopy, let to detect the NO formation, and the efficiency of the process depending on the available amount of free polyphenol, and the intrinsic nature of the hydroxycinnamic acids. Thus, the production of NO through a non-enzymatic mechanism, in light acidic conditions, would account for the antimicrobial action of hydroxycinnamic acids, even in unconventional biological compartments, and for NO as the key-molecule.

Computational studies on nonenzymatic pyroglutamylation mechanism of N-terminal glutamic acid residues in aqueous conditions*

Spontaneous cyclisation of glutamic acid (Glu) residues located at N-termini in peptides and proteins is called ‘pyroglutamylation’ and is assumed to be involved in several neurodegenerative diseases. Although it has long been believed that N-terminal Glu residues undergo pyroglutamylation enzymatically, it has recently been experimentally confirmed that nonenzymatic pyroglutamylation can proceed in some types of aqueous buffer. However, the detailed mechanism has not been proposed or investigated, and even whether some small-molecule catalysts are required for pyroglutamylation has not been clarified. Therefore, we investigated three types of pyroglutamylation mechanism of N-terminal Glu residues using quantum chemical calculations: in the absence of any catalysts, catalysed by one water molecule, and catalysed by two water molecules. All calculations were performed using N-terminal Glu residues capped with a methylamino group on the C-terminal as a model compound. Optimised energy minima and transition state geometries were obtained using the B3LYP density functional method. The pyroglutamylation mechanism is roughly divided into two steps: cyclisation and dehydration, and the calculated activation barrier was 108 and 107 kJ mol−1 in the two- and three-water-assisted pathways, respectively. The results of computational analysis suggest that water molecules can act as catalysts for pyroglutamylation. The calculated activation barrier of two-water-assisted pyroglutamylation was 108 kJ mol−1, and the results of computational analysis indicate that water molecules can act as catalysts for pyroglutamylation.

Physiological and Proteomic Approaches to Address the Active Role of Botrytis cinerea Inoculation in Tomato Postharvest Ripening.

Botrytis cinerea is an unbearable postharvest threat with significant economic impacts. Necrotrophic B. cinerea can readily infect ripe fruit resulting in the rapid progression of symptoms of the disease. To unravel the mechanism by which tomato fruit opposes pathogen attack, we investigated the changes in quality-related attributes as a direct response (DR) or systemic response (SR) of infected tomatoes to the B. cinerea. Additionally, the SR of protein yield and composition were studied in fruit stored at 11 °C/90% relative humidity (RH) for one week. Fungal infection accelerated ripening with increased ethylene and respiration rates. Fruit softening, ascorbic acid and β-carotene increase were associated with DR but not with the SR of the pathogen. Pathogen infection increased lipid peroxidation, causing the production of hydrogen peroxide and oxidative stress, as fruit activated both enzymatic and non-enzymatic mechanisms to trigger stress. B. cinerea increased up to 6.6% the protein yield and downregulated at least 39 proteins. Proteins involved in fruit ripening, such as an ethylene biosynthetic enzyme, were increased in wound-inoculated fruit. Moreover, antioxidant proteins, such as ascorbate peroxidase-APX1 and superoxide dismutase-SOD, increased in infected tomatoes, as these proteins are involved in reactive oxygen species detoxification. Constitutively-expressed proteins tended to be either increased (chaperonin and malate dehydrogenase) or remained unaffected (dehydrin) by pathogen inoculation. Protein levels involved in the metabolism of carbohydrate, the pentose phosphate pathway, terpenoid and flavonoid biosynthesis were differently affected during the treatments. By enabling a better understanding of the fungal direct or systemic response on fruit quality and ripening through biochemical and proteome studies, we may improve the plant–pathogen interaction and complexity.

From Past to Present: The Link Between Reactive Oxygen Species in Sperm and Male Infertility.

Reactive oxygen species (ROS) can be generated in mammalian cells via both enzymatic and non-enzymatic mechanisms. In sperm cells, while ROS may function as signalling molecules for some physiological pathways, the oxidative stress arising from the ubiquitous production of these compounds has been implicated in the pathogenesis of male infertility. In vitro studies have undoubtedly shown that spermatozoa are indeed susceptible to free radicals. However, many reports correlating ROS with sperm function impairment are based on an oxidative stress scenario created in vitro, lacking a more concrete observation of the real capacity of sperm in the production of ROS. Furthermore, sample contamination by leukocytes and the drawbacks of many dyes and techniques used to measure ROS also greatly impact the reliability of most studies in this field. Therefore, in addition to a careful scrutiny of the data already available, many aspects of the relationship between ROS and sperm physiopathology are still in need of further controlled and solid experiments before any definitive conclusions are drawn.

Characterization of the Cofactors Involved in Non-Enzymatic Metmyoglobin Reduction In Vitro

ObjectivesConsumers’ meat purchasing decisions are strongly influenced by color. Myoglobin is the primary meat pigment that contributes to meat color. Myoglobin consists of an iron-containing heme ring and amino acids in the form of globin chains. Both the state of the heme iron and the type of ligand affects meat color. The consumer-preferred bright cherry-red color oxymyoglobin is formed when the iron is in the ferrous state and oxygen bind to the heme. The oxidation of oxymyoglobin or deoxymyoglobin results in the formation of the brown color, ferric metmyoglobin. Predominant metmyoglobin accumulation negatively impacts consumer purchasing choices. Although muscle type and pre- and post-harvest factors can influence meat discoloration, meat has an inherent ability to reduce metmyoglobin through enzymatic pathways, mitochondria-mediated pathways, and non-enzymatic mechanisms. In the enzymatic pathway, an electron from NADH is transferred to metmyoglobin by an enzyme and an electron carrier; while in mitochondria-mediated pathway, an electron from the electron-transport chain is transferred via cytochromes. Previous research speculated the role of non-enzymatic pathway in meat color; however, limited studies have characterized the cofactors present in a meat system. The objectives of this study were to characterize cofactors in non-enzymatic metmyoglobin reduction and determine the effect of storage temperature and postmortem muscle pH in-vitro.Materials and MethodsPurified equine metmyoglobin was reduced in the presence of combinations of electron carriers and donors. Methylene blue and cytochrome c were evaluated as the electron carriers, and NADH and ascorbate were considered as the electron donors. The cofactors were held at 4 and 25°C to determine temperature effects on the reduction of metmyoglobin, and the same cofactor combinations were evaluated at pH of 5.2, 5.6, 6.0, and 6.4 to reflect postmortem muscle pH. Spectrophotometry was utilized to monitor the rates of metmyoglobin reduction. The experiments were replicated five times, and the data were analyzed using the Mixed Procedure of SAS.ResultsThe results indicated that methylene blue was a significantly more effective electron carrier than cytochrome c with both electron donors, ascorbate and NADH. EDTA had no impact on the non-enzymatic metmyoglobin reducing the ability of methylene blue (P = 0.91). Temperature and pH had cofactor specific effects on the non-enzymatic reduction of metmyoglobin. Lower temperature resulted in an increased non-enzymatic metmyoglobin reduction for methylene blue regardless of electron donor (ascorbate, P = 0.03, NADH, P = 0.04). As pH increased, the non-enzymatic metmyoglobin reducing activity reduced significantly in the presence of NADH and methylene blue.ConclusionIn conclusion, the characteristics of the cofactors at specific temperatures and pH impacted the non-enzymatic reduction of metmyoglobin. Further, current in vitro research indicated that non-enzymatic metmyoglobin reduction is possible at lower temperature and meat pH.

Assessment of changes in growth traits, oxidative stress parameters, and enzymatic and non-enzymatic antioxidant defense mechanisms in Lepidium draba plant under osmotic stress induced by polyethylene glycol.

Lepidium draba is a weed with the medicinal properties which few researches have been done on it. In this study, some traits, related to the osmotic stress, in 14-day-old L. draba sprouts that were grown 9 days in the presence of various doses of polyethylene glycol 6000 (PEG 6000) including 0, 3, 6, 9, and 12%, with different osmotic potentials (- 0.04, - 0.12, - 0.23, - 0.34, and - 0.48 MPa, respectively) were investigated. Based on our results, germination percentage besides stem and root lengths decreased with increasing the concentrations of PEG. The contents of electrolyte leakage, malondialdehyde, other aldehydes, total protein, free amino acids, total soluble carbohydrate as well as free proline increased with increasing the concentrations of PEG. Also, for the first time, our results have proven that under osmotic stress, there is an adverse relationship between hydrogen peroxide content and the activity of catalase, peroxidase, ascorbate peroxidase, and guaiacol peroxidase enzymes, such that hydrogen peroxide content decreased with induction of PEG up to 6% and after that increased, while the activity of catalase, peroxidase, ascorbate peroxidase, and guaiacol peroxidase enzymes increased up to 6% PEG and after that decreased. The expression levels of catalase, peroxidase, ascorbate peroxidase, and guaiacol peroxidase genes showed the same pattern as was seen for these enzyme activities. According to the results of this study, it can be deduced that decreasing H2O2 content cannot be the main reason for other oxidative stress parameters to decrease. In this study, P5CS and P5CR gene expression levels increased with increasing levels of PEG up to 12% which was completely similar to free proline content. Based on our results, L. draba can be considered as a semi-tolerant plant to osmotic stress.

Mechanisms of soft tissue and protein preservation in Tyrannosaurus rex

The idea that original soft tissue structures and the native structural proteins comprising them can persist across geological time is controversial, in part because rigorous and testable mechanisms that can occur under natural conditions, resulting in such preservation, have not been well defined. Here, we evaluate two non-enzymatic structural protein crosslinking mechanisms, Fenton chemistry and glycation, for their possible contribution to the preservation of blood vessel structures recovered from the cortical bone of a Tyrannosaurus rex (USNM 555000 [formerly, MOR 555]). We demonstrate the endogeneity of the fossil vessel tissues, as well as the presence of type I collagen in the outermost vessel layers, using imaging, diffraction, spectroscopy, and immunohistochemistry. Then, we use data derived from synchrotron FTIR studies of the T. rex vessels to analyse their crosslink character, with comparison against two non-enzymatic Fenton chemistry- and glycation-treated extant chicken samples. We also provide supporting X-ray microprobe analyses of the chemical state of these fossil tissues to support our conclusion that non-enzymatic crosslinking pathways likely contributed to stabilizing, and thus preserving, these T. rex vessels. Finally, we propose that these stabilizing crosslinks could play a crucial role in the preservation of other microvascular tissues in skeletal elements from the Mesozoic.

Nutritional modulation of the antioxidant capacities in poultry: the case of selenium

Natural antioxidants play important roles in maintaining chicken health, productive and reproductive performance of breeders, layers, rearing birds, and growing broilers. There is a wide range of antioxidant molecules in the body: vitamin E, carotenoids, selenium, ascorbic acid, coenzyme Q, carnitine, taurine, antioxidant enzymes, etc. In the body all antioxidants work together to create the antioxidant network called "antioxidant systems" with Se being the "chief-executive." Analysis of the current data on selenium roles in antioxidant defenses in poultry clearly showed its modulatory effect at the level of breeders, developing embryos, newly hatched chicks, and postnatal chickens. On the one hand, Se is involved in the expression and synthesis of 25 selenoproteins, including GSH-Px, TrxR, and SepP. On the other hand, Se affects non-enzymatic (vitamin E, CoQ, and GSH) and enzymatic (SOD) antioxidant defense mechanisms helping build strong antioxidant defenses. Se efficiency depends on the level of supplementation and form of dietary Se, organic Se sources being more effective modulators of the antioxidant systems in poultry than sodium selenite. Moreover, Se levels in eggs from some wild avian species are close to those found in chicken eggs after 0.3ppm organic Se supplementation and a search for most effective dietary form of organic Se is a priority in poultry nutrition. Antioxidant/prooxidant (redox) balance of the gut and the role/interactions of Se and microbiota in maintaining gut health would be a priority for future poultry research.

Preclinical evaluation of exemestane as a novel chemotherapy for gastric cancer

CYP19A1/aromatase (Ar) is a prognostic biomarker of gastric cancer (GCa). Ar is a critical enzyme for converting androstenedione to oestradiol in the steroidogenesis cascade. For decades, Ar has been targeted with Ar inhibitors (ARIs) in gynaecologic malignancies; however, it is unexplored in GCa. A single‐cohort tissue microarray examination was conducted to study the association between Ar expression and disease outcome in Asian patients with GCa. The results revealed that Ar was a prognostic promoter. Bioinformatics analyses conducted on a Caucasian‐based cDNA microarray databank showed Ar to be positively associated with GCa prognosis for multiple clinical modalities, including surgery, 5‐Fluorouracil (5‐FU) for adjuvant chemotherapy, or HER2 positivity. These findings imply that targeting Ar expression exhibits a potential for fulfilling unmet medical needs. Hence, Ar‐targeting compounds were tested, and the results showed that exemestane exhibited superior cancer‐suppressing efficacy to other ARIs. In addition, exemestane down‐regulated Ar expression. Ablating Ar abundance with short hairpin (sh)Ar could also suppress GCa cell growth, and adding 5‐FU could facilitate this effect. Notably, adding oestradiol could not prevent exemestane or shAr effects, implicating a nonenzymatic mechanism of Ar in cancer growth. Regarding translational research, treatment with exemestane alone exhibited tumour suppression efficacy in a dose‐dependent manner. Combining subminimal doses of 5‐FU and exemestane exerted an excellent tumour suppression effect without influencing bodyweight. This study validated the therapeutic potentials of exemestane in GCa. Combination of metronomic 5‐FU and exemestane for GCa therapy is recommended.

Prenatal Zidovudine Treatment Modifies Early Development of Rat Osteoid \u2013 Confocal Microspectroscopy Analysis

Autofluorescence of the bone extracellular matrix (ECM) has not been widely explored although the ECM plays a very important role in bone development. In our research we focused on examining the bone matrix of very young animals due to the intense growth process during the first month of life. Structure images and fluorescence spectra of the bone surface were carried out using confocal fluorescence microscope Eclipse Ti-S inverted CLSM (NIKON, Japan) for compact tibia of healthy 7-, 14- and 28-day-old rat newborns after prenatal zidovudine administration in comparison with control. Spectral features of ECM autofluorescence were analyzed statistically by taking into consideration p < 0.05. The CLSM technique allows for simultaneous examination of the structure and autofluorescence from selected areas of the bone surface. Excessive autofluorescence of ECM after prenatal zidovudine administration influences bone growth incommensurably to the newborns’ age. Therefore the possibility of an additional non-enzymatic mechanism of collagen cross-linking in the first two weeks of life of newborn rats prenatally treated with zidovudine has been considered. Our results suggest that ECM autofluorescence can be an indicator of bone development in the normal and pathological state.

Jasmonic Acid and K-Phosphite Enhance Productivity and Technological Quality of Sugarcane Crop

Plant resistance inducers are cost-effective and environmentally pleasing strategies of plant protection to mitigate biotic and abiotic agents threatening food safety and energy security. We, accordingly, present jasmonic acid and k-phosphite as low-cost strategies to enhance productive yield and technological quality of sugarcane crop. Exogenously treatment of the sugarcane variety &amp;lsquo;SP81-3250&amp;rsquo; consisted of carrying out foliar application of jasmonic acid at 1, 1.5 and 2 ml L-1 and K-phosphite at 2, 4 and 6 ml L-1 before crop flowering. Interestingly, both systemic phytorregulator and foliar fertilizer at the lowest doses significantly improved contents of total soluble solids and sucrose, as well as productive yield of fermentable sugars. Jasmonic acid could promote growth and development by triggering either non-enzymatic or enzymatic mechanisms in the host self-defense system to support of external stresses, including herbivory by sugarcane-borer. K-phosphite could improve crop performance by not only releasing water-soluble inorganic ions like phosphorus and potash, but also by signaling synthesis of phytoalexins. Purity of cane juice and productivity of stalks sharply decreased, as the doses of the plant-resistance inducers increased. Jasmonic acid and k-phosphite at unbalanced endogenous levels could inhibit physiological ripening and specific enzymatical activity of alkaline invertases and sucrose-phosphate synthase, thereby influencing bioavailability of sucrose. The lower the sucrose content, the poorer the technological quality of sugarcane crop. The conclusion is, therefore, jasmonic acid and K-phosphite at lower doses prove to be effective induced resistance techniques to produce richer feedstock, with potential to produce ethanol fuel and refined sugar in sugar-energy plants.

Uterine non-enzymatic antioxidant defence mechanisms (glutathione, vitamin C, copper and zinc) in diagnosis of canine pyometra.

The aim of the present study was to determine the concentrations of glutathione (GSH), vitamin C, copper (Cu) and zinc (Zn) in the uterine tissues in diagnosis of canine pyometra. Fourteen samples of uterine tissues from female dogs with pyometra and twelve samples of healthy uteruses (control) were used. The concentrations of GSH and vitamin C were determined in the uterine tissue homogenates using spectrophotometric methods. The concentrations of Cu and Zn were measured using atomic absorption spectrometer. The results obtained showed the significantly lower (p⟨0.05) concentration of GSH and the trend towards lower concentration of vitamin C in the pyometra samples compared to the control. The concentrations of Cu and Zn were similar in the uterine tissues from female dogs with pyometra and those from healthy female dogs. The lower GSH and vitamin C concentrations in the uterine tissues of female dogs with pyometra indicate that the non-enzymatic antioxidant mechanisms are impaired in the uterus of dogs with pyometra. These findings suggest that the imbalance of oxidative-antioxidative can play an important role in pathogenesis of canine pyometra.

Unusually High Prevalence of Cosecretion of Ambler Class A and B Carbapenemases and Nonenzymatic Mechanisms in Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa in Lebanon.

The opportunistic pathogen, Pseudomonas aeruginosa, is a main cause of nosocomial infections in Lebanese hospitals. This pathogen is highly threatening due to its ability to develop multiresistance toward a large variety of antibiotics, including the carbapenem subgroup of β-lactams. In this study, we surveyed the enzymatic and nonenzymatic mechanisms of carbapenem resistance in several multidrug-resistant (MDR) strains of P. aeruginosa isolated from patients suffering from nosocomial urinary tract infections in a Lebanese hospital. The occurrence of β-lactamase-encoding genes notably GES, KPC, IMP, VIM, NDM, and OXA, which are characterized by a carbapenemase activity was checked by genomic analyses. Our results provide a first evidence of the occurrence of GES in clinical P. aeruginosa isolates resistant to carbapenems in Lebanon. More interestingly, we showed that almost 40% of the analyzed strains have acquired a dual-carbapenemase secretion of GES-6 and VIM-2 or IMP-15, this being a rare phenomenon among this type of multidrug resistance. Moreover, LC-MS/MS analyses revealed a high prevalence of another enzymatic mechanism of resistance; this is the coexistence of AmpC and Pdc-13 as well as a number of virulence proteins, for instance pilin, lytic transglycosylase, ecotin, chitin-binding protein (Cbp), and TolB-dependent receptor. It is to be noted that a mutation of the oprD2 gene encoding a porin selective for carbapenems has been detected in almost 66% of our strains. All in all, our study reveals by the use of different methods, unusual simultaneous enzymatic (GES, IMP, VIM, pdc13, and AmpC) and nonenzymatic mechanisms of resistance (reduction of OprD2 expression) for MDR Pseudomonas aeruginosa.

The ETS Inhibitors YK-4-279 and TK-216 Are Novel Antilymphoma Agents.

Transcription factors are commonly deregulated in cancer, and they have been widely considered as difficult to target due to their nonenzymatic mechanism of action. Altered expression levels of members of the ETS-transcription factors are often observed in many different tumors, including lymphomas. Here, we characterized two small molecules, YK-4-279 and its clinical derivative, TK-216, targeting ETS factors via blocking the protein-protein interaction with RNA helicases, for their antilymphoma activity. The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination; validation experiments on in vivo models; and transcriptome and coimmunoprecipitation experiments. YK-4-279 and TK-216 demonstrated an antitumor activity across several lymphoma cell lines, which we validated in vivo. We observed synergistic activity when YK-4-279 and TK-216 were combined with the BCL2 inhibitor venetoclax and with the immunomodulatory drug lenalidomide. YK-4-279 and TK-216 interfere with protein interactions of ETS family members SPIB, in activated B-cell-like type diffuse large B-cell lymphomas, and SPI1, in germinal center B-cell-type diffuse large B-cell lymphomas. The ETS inhibitor YK-4-279 and its clinical derivative TK-216 represent a new class of agents with in vitro and in vivo antitumor activity in lymphomas. Although their detailed mechanism of action needs to be fully defined, in DLBCL they might act by targeting subtype-specific essential transcription factors.