In Silico Design of a Chimeric Humanized L-asparaginase.

Acute lymphoblastic leukemia (ALL) is the most common cancer among children worldwide, characterized by an overproduction of undifferentiated lymphoblasts in the bone marrow. The treatment of choice for this disease is the enzyme L-asparaginase (ASNase) from bacterial sources. ASNase hydrolyzes circulating L-asparagine in plasma, leading to starvation of leukemic cells. The ASNase formulations of E. coli and E. chrysanthemi present notorious adverse effects, especially the immunogenicity they generate, which undermine both their effectiveness as drugs and patient safety. In this study, we developed a humanized chimeric enzyme from E. coli L-asparaginase which would reduce the immunological problems associated with current L-asparaginase therapy. For these, the immunogenic epitopes of E. coli L-asparaginase (PDB: 3ECA) were determined and replaced with those of the less immunogenic Homo sapiens asparaginase (PDB:4O0H). The structures were modeled using the Pymol software and the chimeric enzyme was modeled using the SWISS-MODEL service. A humanized chimeric enzyme with four subunits similar to the template structure was obtained, and the presence of asparaginase enzymatic activity was predicted by protein-ligand docking.

Helicobacter pyloril-asparaginase: a study of immunogenicity from an in silico approach.

Helicobacter pylori has become the causal agent of multiple forms of gastric disease worldwide, including gastric cancer. The enzyme l-asparaginase (ASNase) has been studied as a virulence factor. In this work, we performed an in silico investigation to characterize the immunological profile of H. pylori ASNase (HpASNase) to ascertain the possible implication of HpASNase immunogenicity in the H. pylori virulence mechanism. We applied a workflow based on bioinformatics tools, which, by calculating the relative frequency of immunogenic T-cell and B-cell epitopes, allowed us to predict the immunogenicity and allergenicity of HpASNase in silico. We also visualized the epitopes by mapping them into the native structure of the enzyme. We report for the first time the T-cell and B-cell epitope composition that contributes to the immunogenicity of this HpASNase, as well as the regions that could generate a hypersensitivity response in humans. ASNase from H. pylori resulted in highly immunogenic and allergenic. The high immunogenicity of HpASNase could imply the pathogenic mechanisms of H. pylori. This knowledge could be important for the development of new drugs against H. pylori infections. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03359-0.

Efectos biológicos de la exposición a distintas concentraciones de oxígeno: desde la hipoxia hipobárica al oxígeno hiperbárico / Biological effects due to exposure to different concentrations of oxygen from hypo to hyperoxemia

The systemic effects of oxygen deficiency or excess are not thoroughly described. Knowledge is evolving towards the description of beneficial and detrimental effects of both extremes of partial pressure of oxygen (PaO2). The cellular and tissue mediators derived from the modulation of the oxidative tone and the production of reactive oxygen species (ROS) are widely characterized biochemically, but the pathophysiological characterization is lacking. Preclinical models support the use of hypobaric hypoxia preconditioning, based on its beneficial effects on ventricular function or its reduction in infarct size. A very important use of oxygen today is in commercial diving. However, novel clinical indications for oxygen such as the healing of diabetic foot ulcers and bone injury caused by radiotherapy are increasingly used. On the other hand, the modulation of the hypoxic response associated with exposure to high altitude environments (hypobaric), favors Chile and its highlands as a natural laboratory to determine certain cardiovascular, cerebral and metabolic responses in the resident population. Also, the consequences of the intermittent exposure to high altitudes in workers also deserves attention. This review discusses the physiopathological response to hypo and hyperoxemia, associated with environments with different oxygen concentrations, and brings back the concept of oxygen as a pharmacological mediator in extreme environments such as high altitudes and hyperbaric medicine in divers, decompression sickness, osteonecrosis associated with radiotherapy and sudden sensorineural hearing loss.

[Biological effects due to exposure to different concentrations of oxygen from hypo to hyperoxemia]. / Efectos biológicos de la exposición a distintas concentraciones de oxígeno: desde la hipoxia hipobárica al oxígeno hiperbárico.

The systemic effects of oxygen deficiency or excess are not thoroughly described. Knowledge is evolving towards the description of beneficial and detrimental effects of both extremes of partial pressure of oxygen (PaO2). The cellular and tissue mediators derived from the modulation of the oxidative tone and the production of reactive oxygen species (ROS) are widely characterized biochemically, but the pathophysiological characterization is lacking. Preclinical models support the use of hypobaric hypoxia preconditioning, based on its beneficial effects on ventricular function or its reduction in infarct size. A very important use of oxygen today is in commercial diving. However, novel clinical indications for oxygen such as the healing of diabetic foot ulcers and bone injury caused by radiotherapy are increasingly used. On the other hand, the modulation of the hypoxic response associated with exposure to high altitude environments (hypobaric), favors Chile and its highlands as a natural laboratory to determine certain cardiovascular, cerebral and metabolic responses in the resident population. Also, the consequences of the intermittent exposure to high altitudes in workers also deserves attention. This review discusses the physiopathological response to hypo and hyperoxemia, associated with environments with different oxygen concentrations, and brings back the concept of oxygen as a pharmacological mediator in extreme environments such as high altitudes and hyperbaric medicine in divers, decompression sickness, osteonecrosis associated with radiotherapy and sudden sensorineural hearing loss.

Role of Oxidative Stress in the Mechanisms of Anthracycline-Induced Cardiotoxicity: Effects of Preventive Strategies.

Anthracycline-induced cardiotoxicity (AIC) persists as a significant cause of morbidity and mortality in cancer survivors. Although many protective strategies have been evaluated, cardiotoxicity remains an ongoing threat. The mechanisms of AIC remain unclear; however, several pathways have been proposed, suggesting a multifactorial origin. When the central role of topoisomerase 2ß in the pathophysiology of AIC was described some years ago, the classical reactive oxygen species (ROS) hypothesis shifted to a secondary position. However, new insights have reemphasized the importance of the role of oxidative stress-mediated signaling as a common pathway and a critical modulator of the different mechanisms involved in AIC. A better understanding of the mechanisms of cardiotoxicity is crucial for the development of treatment strategies. It has been suggested that the available therapeutic interventions for AIC could act on the modulation of oxidative balance, leading to a reduction in oxidative stress injury. These indirect antioxidant effects make them an option for the primary prevention of AIC. In this review, our objective is to provide an update of the accumulated knowledge on the role of oxidative stress in AIC and the modulation of the redox balance by potential preventive strategies.

Antenatal melatonin modulates an enhanced antioxidant/pro-oxidant ratio in pulmonary hypertensive newborn sheep.

Chronic hypobaric hypoxia during fetal and neonatal life induces neonatal pulmonary hypertension. Hypoxia and oxidative stress are driving this condition, which implies an increase generation of reactive oxygen species (ROS) and/or decreased antioxidant capacity. Melatonin has antioxidant properties that decrease oxidative stress and improves pulmonary vascular function when administered postnatally. However, the effects of an antenatal treatment with melatonin in the neonatal pulmonary function and oxidative status are unknown. Therefore, we hypothesized that an antenatal therapy with melatonin improves the pulmonary arterial pressure and antioxidant status in high altitude pulmonary hypertensive neonates. Twelve ewes were bred at high altitude (3600 m); 6 of them were used as a control group (vehicle 1.4% ethanol) and 6 as a melatonin treated group (10 mg d-1 melatonin in vehicle). Treatments were given once daily during the last third of gestation (100-150 days). Lambs were born and raised with their mothers until 12 days old, and neonatal pulmonary arterial pressure and resistance, plasma antioxidant capacity and the lung oxidative status were determined. Furthermore, we measured the pulmonary expression and activity for the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase, and the oxidative stress markers 8-isoprostanes, 4HNE and nitrotyrosine. Finally, we assessed pulmonary pro-oxidant sources by the expression and function of NADPH oxidase, mitochondria and xanthine oxidase. Melatonin decreased the birth weight. However, melatonin enhanced the plasma antioxidant capacity and decreased the pulmonary antioxidant activity, associated with a diminished oxidative stress during postnatal life. Interestingly, melatonin also decreased ROS generation at the main pro-oxidant sources. Our findings suggest that antenatal administration of melatonin programs an enhanced antioxidant/pro-oxidant status, modulating ROS sources in the postnatal lung.

From Synthesis to Characterization of Site-Selective PEGylated Proteins.

Covalent attachment of therapeutic proteins to polyethylene glycol (PEG) is widely used for the improvement of its pharmacokinetic and pharmacological properties, as well as the reduction in reactogenicity and related side effects. This technique named PEGylation has been successfully employed in several approved drugs to treat various diseases, even cancer. Some methods have been developed to obtain PEGylated proteins, both in multiple protein sites or in a selected amino acid residue. This review focuses mainly on traditional and novel examples of chemical and enzymatic methods for site-selective PEGylation, emphasizing in N-terminal PEGylation, that make it possible to obtain products with a high degree of homogeneity and preserve bioactivity. In addition, the main assay methods that can be applied for the characterization of PEGylated molecules in complex biological samples are also summarized in this paper.

Antinociception induced by rosuvastatin in murine neuropathic pain.

BACKGROUND: Neuropathic pain, and subsequent hypernociception, can be induced in mice by paclitaxel (PTX) administration and partial sciatic nerve ligation (PSNL). Its pharmacotherapy has been a clinical challenge, due to a lack of effective treatment. In two models of mouse neuropathic pain (PTX and PSNL) the antinociception induced by rosuvastatin and the participation of proinflammatory biomarkers, interleukin (IL)- 1ß, TBARS and glutathione were evaluated. METHODS: A dose-response curve for rosuvastatin ip was obtained on cold plate, hot plate and Von Frey assays. Changes on spinal cord levels of IL-1ß, glutathione and lipid peroxidation were measured at 7 and 14days in PTX and PSNL murine models. RESULTS: PTX or PSNL were able to induce in mice peripheral neuropathy with hypernociception, either to 7 and 14days. Rosuvastatin induced a dose dependent antinociception in hot plate, cold plate and Von Frey assays. The increased levels of IL-1ß or TBARS induced by pretreatment with PTX or PSNL were reduced by rosuvastatin. The reduction of spinal cord glutathione, by PTX or PSNL, expressed as the ratio GSH/GSSG, were increased significantly in animals pretreated with rosuvastatin. The anti-inflammatory properties of statins could underlie their beneficial effects on neuropathic pain by reduction of proinflammatory biomarkers and activation of glia. CONCLUSION: The findings of this study suggest a potential usefulness of rosuvastatin in the treatment of neuropathic pain.

Molecular aspects of breast cancer resistance to drugs (Review).

Despite continuous advances in the knowledge of breast cancer pathophysiology, this type of neoplasia remains a leading cause of cancer-related death in women worldwide. Carcinogenesis takes a progressive course from somatic mutations, alteration of the DNA repair mechanisms, inhibition of growth suppressors, followed by cell proliferation, tissue invasion and risk of metastasis. Less than 10% of all cancers are hereditary, and in the case of breast cancer only 8%, a phenomenon linked to genetic changes in BRCA1 or BRCA2. All the other cancers can be caused by an infection (15%) or in most cases (75%) the etiology is unknown. Patients with genetic mutations in BRCA1 or BRCA2 have 30-60% likelihood of developing a second primary breast cancer and between 11 and 45% risk of ovarian cancer, HER-2/neu is overexpressed in ~30% of human breast tumors and it has a predictive role in chemotherapy and endocrine therapy.

Ω3 Supplementation and intermittent hypobaric hypoxia induce cardioprotection enhancing antioxidant mechanisms in adult rats.

Intermittent hypobaric hypoxia (IH) is linked with oxidative stress, impairing cardiac function. However, early IH also activate cardio-protective mechanisms. Omega 3 fatty acids (Ω3) induce cardioprotection by reducing infarct size and reinforcing antioxidant defenses. The aim of this work was to determine the combined effects of IH and Ω3 on cardiac function; oxidative balance and inflammatory state. Twenty-eight rats were randomly divided into four groups: normobaric normoxia (N); N + Ω3 (0.3 g·kg-1·day-1); IH; and IH + Ω3. IH was induced by 4 intercalate periods of hypoxia (4 days)-normoxia (4 days) in a hypobaric chamber during 32 days. At the end of the exposure, hearts were mounted in a Langendorff system and subjected to 30 min of ischemia followed by 120 min of reperfusion. In addition, we determined HIF-1α and ATP levels, as well as oxidative stress by malondialdehyde and nitrotyrosine quantification. Further, the expression of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase was determined. NF-kappaB and myeloperoxidase levels were assessed in the hearts. Relative to N hearts, IH improved left ventricular function (Left ventricular developed pressure: N; 21.8 ± 3.4 vs. IH; 42.8 ± 7.1 mmHg; p < 0.05); reduced oxidative stress (Malondialdehyde: N; 14.4 ± 1.8 vs. IH; 7.3 ± 2.1 µmol/mg prot.; p < 0.05); and increased antioxidant enzymes expression. Supplementation with Ω3 induces similar responses as IH group. Our findings suggest that both, IH and Ω3 in an independent manner, induce functional improvement by antioxidant and anti-inflammatory mechanisms, establishing cardio-protection.

Omega 3 chronic supplementation attenuates myocardial ischaemia-reperfusion injury through reinforcement of antioxidant defense system in rats.

Currently, controversial clinical data about the protective effects in the consumption of n-3 polyunsaturated fatty acids (PUFAs) in ischaemic heart diseases exist. Improved myocardial resistance to ischaemia-reperfusion (IR) injury results in non-lethal myocardial infarction, which is a relevant factor in the myocardial function. We hypothesized that chronic supplementation with PUFAs reduced infarct size (IS) and induced an improvement on oxidative stress-related parameters in IR model. Rats were supplemented with two doses of PUFAs D1 (n = 7) (0.6 g kg(-1) d(-1) ) and D2 (n = 7) (1.2 g kg(-1) d(-1) ) for 8 weeks. Control group (n = 7) received only standard diet. In ex vivo model, all rat hearts were subjected to 30 min of global ischaemia followed by 120 min of reperfusion. The IS and left ventricular function were assessed. Lipid peroxidation, reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio and antioxidant enzyme activity were measured in the whole heart. The results show a reduction in IS in a dose-dependent manner with PUFAs D1 (30.6%) and D2 (48.5%) and higher values of left ventricular developed pressure, at the end of the reperfusion, for each dose, respectively (p < 0.05). The two PUFAs groups showed higher values of GSH/GSSG ratio and lipid peroxidation, and higher values of activity of antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase at basal condition (p < 0.05). At the end of reperfusion, the GSH/GSSG ratio and antioxidants enzyme activity did not show a significant drop in their values (p > 0.05). These findings suggested that the supplementation with PUFAs induces cardioprotection against IR injury, associated with reinforcement of the antioxidant defense system.

Relationship between severity of adult community-acquired pneumonia and impairment of the antioxidant defense system.

Oxidant/antioxidant imbalance has been reported in some infectious diseases, including community-acquired pneumonia (CAP). The aim was to assess the antioxidant status in adults with CAP and its relationship with clinical severity at admission. Fifty-nine patients with CAP were enrolled and categorized at admission by the FINE score, from July 2010 to October 2012. In the same period 61 controls were enrolled. Plasma samples were obtained at admission for determination of the ferric reducing ability of plasma (FRAP) and lipid peroxidation (8-isoprostane). Erythrocyte reduced (GSH)/oxidized (GSSG) glutathione, malondialdehyde (MDA) and antioxidant enzyme activity were assessed. Antioxidant status in adults with CAP represented by FRAP and the GSH/GSSG ratio were 16.8% (p=0.03) and 39.7% (p=0.04) lower than control values, respectively. In addition, FRAP values showed a positive correlation with GSH/GSSG ratio (r=0.852; p<0.02; n=59). The CAP group showed greater lipid peroxidation in both plasma and erythrocytes. The FINE score correlated negatively with FRAP (r= -0.718; p<0.05; n=59) and positively with MDA and F2 isoprostane levels (r=0.673; p<0.05; n=59; r=0.892; p<0.01; n=59, respectively). Antioxidant status alterations correlated with clinical severity. The FRAP assay and lipid peroxidation biomarkers may provide a useful parameter for estimating the severity and the clinical outcome of patients with CAP.

Cellular and molecular mechanisms in the hypoxic tissue: role of HIF-1 and ROS.

Reactive oxygen species such as superoxide anion radicals (O2 (-) ) and hydrogen peroxide (H2 O2 ) have for long time been recognized as undesirable by-products of the oxidative mitochondrial generation of adenosine triphosphate (ATP). Recently, these highly reactive species have been associated to important signaling pathways in diverse physiological conditions such as those activated in hypoxic microenvironments. The molecular response to hypoxia requires fast-acting mechanisms acting within a wide range of partial pressures of oxygen (O2 ). Intracellular O2 sensing is an evolutionary preserved feature, and the best characterized molecular responses to hypoxia are mediated through transcriptional activation. The transcription factor, hypoxia-inducible factor 1 (HIF-1), is a critical mediator of these adaptive responses, and its activation by hypoxia involves O2 -dependent posttranslational modifications and nuclear translocation. Through the induction of the expression of its target genes, HIF-1 coordinately regulates tissue O2 supply and energetic metabolism. Other transcription factors such as nuclear factor κB are also redox sensitive and are activated in pro-oxidant and hypoxic conditions. The purpose of this review is to summarize new developments in HIF-mediated O2 sensing mechanisms and their interactions with reactive oxygen species-generating pathways in normal and abnormal physiology.

Relationship between severity of adult community-acquired pneumonia and impairment of the antioxidant defense system

Oxidant/antioxidant imbalance has been reported in some infectious diseases, including community-acquired pneumonia (CAP). The aim was to assess the antioxidant status in adults with CAP and its relationship with clinical severity at admission. Fifty-nine patients with CAP were enrolled and categorized at admission by the FINE score, from July 2010 to October 2012. In the same period 61 controls were enrolled. Plasma samples were obtained at admission for determination of the ferric reducing ability of plasma (FRAP) and lipid peroxidation (8-isoprostane). Erythrocyte reduced (GSH)/oxidized (GSSG) glutathione, malondialdehyde (MDA) and antioxidant enzyme activity were assessed. Antioxidant status in adults with CAP represented by FRAP and the GSH/GSSG ratio were 16.8% (p=0.03) and 39.7% (p=0.04) lower than control values, respectively. In addition, FRAP values showed a positive correlation with GSH/GSSG ratio (r=0.852; p<0.02; n=59). The CAP group showed greater lipid peroxidation in both plasma and erythrocytes. The FINE score correlated negatively with FRAP (r= -0.718; p<0.05; n=59) and positively with MDA and F2 isoprostane levels (r=0.673; p<0.05; n=59; r=0.892; p<0.01; n=59, respectively). Antioxidant status alterations correlated with clinical severity. The FRAP assay and lipid peroxidation biomarkers may provide a useful parameter for estimating the severity and the clinical outcome of patients with CAP.