The enzymes of the oxidative phase of the pentose phosphate pathway as targets of reactive species: consequences for NADPH production.

The pentose phosphate pathway (PPP) is a key metabolic pathway. The oxidative phase of this process involves three reactions catalyzed by glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL) and 6-phosphogluconate dehydrogenase (6PGDH) enzymes. The first and third steps (catalyzed by G6PDH and 6PGDH, respectively) are responsible for generating reduced nicotinamide adenine dinucleotide phosphate (NAPDH), a key cofactor for maintaining the reducing power of cells and detoxification of both endogenous and exogenous oxidants and electrophiles. Despite the importance of these enzymes, little attention has been paid to the fact that these proteins are targets of oxidants. In response to oxidative stimuli metabolic pathways are modulated, with the PPP often up-regulated in order to enhance or maintain the reductive capacity of cells. Under such circumstances, oxidation and inactivation of the PPP enzymes could be detrimental. Damage to the PPP enzymes may result in a downward spiral, as depending on the extent and sites of modification, these alterations may result in a loss of enzymatic activity and therefore increased oxidative damage due to NADPH depletion. In recent years, it has become evident that the three enzymes of the oxidative phase of the PPP have different susceptibilities to inactivation on exposure to different oxidants. In this review, we discuss existing knowledge on the role that these enzymes play in the metabolism of cells, and their susceptibility to oxidation and inactivation with special emphasis on NADPH production. Perspectives on achieving a better understanding of the molecular basis of the oxidation these enzymes within cellular environments are given.

Antioxidant Capacity of Free and Peptide Tryptophan Residues Determined by the ORAC (Oxygen Radical Absorbance Capacity) Assay Is Modulated by Radical-Radical Reactions and Oxidation Products.

The ORAC (Oxygen Radical Absorbance Capacity) assay is commonly employed for determining the antioxidant capacity of bioactive peptides. To gain insights into the meaning of this index for peptides containing a single Trp, we studied the consumption of this residue and fluorescein (FLH, the probe of ORAC method), induced by radicals generated by AAPH (2,2'-Azo-bis(2-amidinopropane) dihydrochloride) thermolysis. ORAC values were rationalized from kinetics and computational calculations of bond dissociation energies (BDE) of the N-H bond (indole ring of Trp). Free Trp, di- and tri- peptides, and three larger peptides were studied. Solutions containing 70 nM FLH, 1-5 µM free Trp or peptides, and 10 mM AAPH were incubated at 37 °C in phosphate buffer. Kinetic studies showed that FLH minimally affected Trp consumption. However, a clear protection of FLH, characterized by pseudo-lag times, was evidenced, reflecting radical-radical reactions and FLH repairing. Peptides showed similar ORAC values (~1.9-2.8 Trolox equivalents), while BDE varied between 91.9 and 103.5 kcal. These results, added to the protection of FLH observed after total consumption of Trp, indicate a lack of discrimination of the assay for the chemical structure of peptides and the contribution of oxidation products to the index.

Implications of differential peroxyl radical-induced inactivation of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase for the pentose phosphate pathway.

Escherichia coli glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are key enzymes of the pentose phosphate pathway, responsible for the NADPH production in cells. We investigated modification of both enzymes mediated by peroxyl radicals (ROO·) to determine their respective susceptibilities to and mechanisms of oxidation. G6PDH and 6PGDH were incubated with AAPH (2,2'-azobis(2-methylpropionamidine)dihydrochloride), which was employed as ROO· source. The enzymatic activities of both enzymes were determined by NADPH release, with oxidative modifications examined by electrophoresis and liquid chromatography (LC) with fluorescence and mass (MS) detection. The activity of G6PDH decreased up to 62.0 ± 15.0% after 180 min incubation with 100 mM AAPH, whilst almost total inactivation of 6PGDH was determined under the same conditions. Although both proteins contain abundant Tyr (particularly 6PGDH), these residues were minimally affected by ROO·, with Trp and Met being major targets. LC-MS and in silico analysis showed that the modification sites of G6PDH are distant to the active site, consistent with a dispersed distribution of modifications, and inactivation resulting from oxidation of multiple Trp and Met residues. In contrast, the sites of oxidation detected on 6PGDH are located close to its catalytic site indicating a more localized oxidation, and a consequent high susceptibility to ROO·-mediated inactivation.

Role of amino acid oxidation and protein unfolding in peroxyl radical and peroxynitrite-induced inactivation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

The mechanisms underlying the inactivation of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PDH) induced by peroxyl radicals (ROO●) and peroxynitrite (ONOO-), were explored. G6PDH was incubated with AAPH (2,2' -azobis(2-methylpropionamidine)dihydrochloride), used as ROO● source, and ONOO-. Enzymatic activity was assessed by NADPH generation, while oxidative modifications were analyzed by gel electrophoresis and liquid chromatography (LC) with fluorescence and mass detection. Changes in protein conformation were studied by circular dichroism (CD) and binding of the fluorescent dye ANS (1-anilinonaphthalene-8-sulfonic acid). Incubation of G6PDH (54.4 µM) with 60 mM AAPH showed an initial phase without significant changes in enzymatic activity, followed by a secondary time-dependent continuous decrease in activity to ∼59% of the initial level after 90 min. ONOO- induced a significant and concentration-dependent loss of G6PDH activity with ∼46% of the initial activity lost on treatment with 1.5 mM ONOO-. CD and ANS fluorescence indicated changes in G6PDH secondary structure with exposure of hydrophobic sites on exposure to ROO●, but not ONOO-. LC-MS analysis provided evidence for ONOO--mediated oxidation of Tyr, Met and Trp residues, with damage to critical Met and Tyr residues underlying enzyme inactivation, but without effects on the native (dimeric) state of the protein. In contrast, studies using chloramine T, a specific oxidant of Met, provided evidence that oxidation of specific Met and Trp residues and concomitant protein unfolding, loss of dimer structure and protein aggregation are involved in G6PDH inactivation by ROO●. These two oxidant systems therefore have markedly different effects on G6PDH structure and activity.

Integrated photothermal decontamination device for N95 respirators.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the COVID-19 global pandemic has infected over 25 million people worldwide and resulted in the death of millions. The COVID-19 pandemic has also resulted in a shortage of personal protective equipment (PPE) in many regions around the world, particularly in middle- and low-income countries. The shortages of PPE, such as N95 respirators, is something that will persist until an effective vaccine is made available. Thus, devices that while being easy to operate can also be rapidly deployed in health centers, and long-term residences without the need for major structural overhaul are instrumental to sustainably use N95 respirators. In this report, we present the design and validation of a decontamination device that combines UV-C & B irradiation with mild-temperature treatment. The device can decontaminate up to 20 masks in a cycle of < 30 min. The decontamination process did not damage or reduce the filtering capacity of the masks. Further, the efficacy of the device to eliminate microbes and viruses from the masks was also evaluated. The photothermal treatment of our device was capable of eradicating > 99.9999% of the bacteria and > 99.99% of the virus tested.

Azocompounds as generators of defined radical species: Contributions and challenges for free radical research.

Peroxyl radicals participate in multiple processes involved in critical changes to cells, tissues, pharmacueticals and foods. Some of these reactions explain their association with degenerative pathologies, including cardiovascular and neurological diseases, as well as cancer development. Azocompounds, and particularly AAPH (2,2'-Azobis(2-methylpropionamidine) dihydrochloride), a cationic water-soluble derivative, have been employed extensively as sources of model peroxyl radicals. A considerable number of studies have reported mechanistic data on the oxidation of biologically-relevant targets, the scavenging activity of foods and natural products, and the reactions with, and responses of, cultured cells. However, despite the (supposed) experimental simplicity of using azocompounds, the chemistry of peroxyl radical production and subsequent reactions is complicated, and not always considered in sufficient depth when analyzing experimental data. The present work discusses the chemical aspects of azocompounds as generators of peroxyl (and other) radicals, together with their contribution to our understanding of biochemistry, pharmaceutical and food chemistry research. The evidence supporting a role for the formation of alkoxyl (RO•) and other radicals during thermal and photochemical decomposition of azocompounds is assessed, together with the potential influence of such species on the reactions under study.

Protein quantification by bicinchoninic acid (BCA) assay follows complex kinetics and can be performed at short incubation times.

Amongst the available methodologies for protein determination, the bicinchoninic acid (BCA) assay highlights for its simplicity, sensitivity, repeatability and reproducibility. Nevertheless, in spite that the general principle behind this methodology is known, there are still unanswered questions regarding the chemistry behind the assay and the experimental conditions commonly employed. The present work explored the kinetics, and the analytical response of the assay to free amino acids, peptides (containing tryptophan and tyrosine), and proteins. Results revealed kinetic profiles characterized by the absence of plateaus, with behaviors depending on the type of the sample. The latter, along with contribution to the BCA index elicited by oxidation products generated at the side chain of tryptophan and tyrosine, as well as pre-oxidized ß-casein, evidenced the presence of complex reaction mechanisms. In spite of such complexity, our results showed that the BCA index is not modulated by the incubation time. This applies for responses producing absorbance intensities (at 562 nm) higher than 0.1. Therefore, we propose that the assay can be applied at shorter incubation times (15 min) than those indicated in manufactures specifications, and usually used by researches and industry (30 min at 37 °C).

Formation and characterization of crosslinks, including Tyr-Trp species, on one electron oxidation of free Tyr and Trp residues by carbonate radical anion.

Dityrosine and ditryptophan bonds have been implied in protein crosslinking. This is associated with oxidative stress conditions including those involved in neurodegenerative pathologies and age-related processes. Formation of dityrosine and ditryptophan derives from radical-radical reactions involving Tyr˙ and Trp˙ radicals. However, cross reactions of Tyr˙ and Trp˙ leading to Tyr-Trp crosslinks and their biological consequences have been less explored. In the present work we hypothesized that exposure of free Tyr and Trp to a high concentration of carbonate anion radicals (CO3˙-), under anaerobic conditions, would result in the formation of Tyr-Trp species, as well as dityrosine and ditryptophan crosslinks. Here we report a simple experimental procedure, employing CO3˙- generated photochemically by illumination of a Co(iii) complex at 254 nm, that produces micromolar concentrations of Tyr-Trp crosslinks. Analysis by mass spectrometry of solutions containing only the individual amino acids, and the Co(iii) complex, provided evidence for the formation of o,o'-dityrosine and isodityrosine from Tyr, and three ditryptophan dimers from Trp. When mixtures of Tyr and Trp were illuminated in an identical manner, Tyr-Trp crosslinks were detected together with dityrosine and ditryptophan dimers. These results indicate that there is a balance between the formation of these three classes of crosslinks, which is dependent on the Tyr and Trp concentrations. The methods reported here allow the generation of significant yields of isolated Tyr-Trp adducts and their characterization. This technology should facilitate the detection, and examination of the biological consequences of Tyr-Trp crosslink formation in complex systems in future investigations.

Quantification of carbonate radical formation by the bicarbonate-dependent peroxidase activity of superoxide dismutase 1 using pyrogallol red bleaching.

Carbonate radicals (CO3-) are generated by the bicarbonate-dependent peroxidase activity of cytosolic superoxide dismutase (Cu,Zn-SOD, SOD-1). The present work explored the use of bleaching of pyrogallol red (PGR) dye to quantify the rate of CO3- formation from bovine and human SOD-1 (bSOD-1 and hSOD-1, respectively). This approach was compared to previously reported methods using electron paramagnetic resonance spin trapping with DMPO, and the oxidation of ABTS (2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid). The kinetics of PGR consumption elicited by CO3- was followed by visible spectrophotometry. Solutions containing PGR (5-200 µM), SOD-1 (0.3-3 µM), H2O2 (2 mM) in bicarbonate buffer (200 mM, pH 7.4) showed a rapid loss of the PGR absorption band centered at 540 nm. The initial consumption rate (Ri) gave values independent of the initial PGR concentration allowing an estimate to be made of the rate of CO3- release of 24.6 ±â€¯4.3 µM min-1 for 3 µM bSOD-1. Both bSOD-1 and hSOD-1 showed a similar peroxidase activity, with enzymatic inactivation occurring over a period of 20 min. The single Trp residue (Trp32) present in hSOD-1 was rapidly consumed (initial consumption rate 1.2 ±â€¯0.1 µM min-1) with this occurring more rapidly than hSOD-1 inactivation, suggesting that these processes are not directly related. Added free Trp was rapidly oxidized in competition with PGR. These data indicate that PGR reacts rapidly and efficiently with CO3- resulting from the peroxidase activity of SOD-1, and that PGR-bleaching is a simple, fast and cheap method to quantify CO3- release from bSOD-1 and hSOD-1 peroxidase activity.

Principios de cirugía oncológica / Principles of surgical oncology

La cirugía oncológica se desarrolló como una rama de la cirugía general, que busca facilitar la interacción entre la radioterapia, la oncología médica y las demás especialidades, para concertar un plan terapéutico integral y multidisciplinario del paciente con cáncer. En este artículo se presenta una revisión narrativa de los principios básicos del abordaje diagnóstico y terapéutico del paciente oncológico, bien sea con un diagnóstico inicial o recurrente. Se hace particular énfasis en la necesidad de plantear un tratamiento curativo de ser posible, con márgenes claros y con una aproximación quirúrgica que tenga en cuenta el comportamiento y la diseminación tumoral. Cuando la curación no sea posible, en el contexto de un tratamiento multimodal, la cirugía paliativa es una opción válida y acertada. Los principios de la cirugía oncológica deben ser interiorizados y respetados por todos los cirujanos que enfrentan y tratan pacientes con cáncer. La observancia de estos principios garantizará mejores resultados para los pacientes.

Surgical Oncology has developed as the branch of general surgery that seeks to facilitate the interaction between different specialties, such as medical oncology, radiation oncology and many others, in order to offer a complete and multidisciplinary management of the patient with cancer. In this article we present a narrative review of the basic principles for the diagnosis and therapeutic approach in the oncology patient, either in patients with new diagnosis or in those with recurrent disease. We made particular emphasis on the need to formulate a curative strategy, with clear and negative margins and taking into consideration the pattern of oncologic spread and biologic behavior. In cases where cure is not feasible, and in the context of a multimodal approach, palliative surgery is a valid and appropriate option. The basic principles of surgical oncology should be adopted and respected by all surgeons that treat patients with cancer. The proper adherence to these principles will guarantee better results for these patients.

Prevalence of BRCA1 and BRCA2 mutations in unselected breast cancer patients from medellín, Colombia.

BACKGROUND: Approximately 5% of all breast cancers can be attributed to a mutation in the BRCA1 or BRCA2 gene. The genetic component of breast cancer in Colombia has been, for the most part, studied on cases from the Bogota region. Five different founder mutations were in two studies of breast cancer patients in the Bogota region. It is important that the frequency of mutations be established among unselected cases of breast cancer of other regions of Colombia in order to estimate the genetic burden of this cancer in Colombia and to plan genetic services. The aim of this study was to establish the mutation frequencies of the BRCA genes in breast cancer patients unselected for family history or age, from Medellin, Colombia. METHODS: We enrolled 280 unselected women with breast cancer from a large public hospital in Medellin, Colombia. A detailed family history from each patient and a blood sample was obtained and processed for DNA analysis. Mutations in BRCA1 and BRCA2 were sought using a combination of techniques including a panel of recurrent Hispanic BRCA mutations which consists of fifty BRCA1 mutations and forty-six BRCA2 mutations, including the five recurrent Colombian BRCA mutations. All mutations were confirmed by direct sequencing. RESULTS: Genetic testing was successfully completed for 244 of the 280 cases (87%). Among the 244 cases, three deleterious mutations were identified (two in BRCA1 and one in BRCA2) representing 1.2% of the total. The average age of breast cancer in the mutation-positive cases was 34 years. The two BRCA1 mutations were known founder mutations (3450del4 in exon 11 and A1708E in exon 18). The BRCA2 mutation was in exon 11 (5844del5) and has not been previously reported in individuals of Colombian descent. Among the three mutation-positive families was a breast cancer family and two families with no history of breast or ovarian cancer. CONCLUSION: The frequency of BRCA mutations in unselected breast cancer cases from the Medellin region of Colombia is low and is approximately 1.2%.

Descripción de dos abordajes quirúrgicos para tumores retroperitoneales, experiencia en el Hospital Pablo Tobón Uribe, 2003-2011 / Description of two surgical approaches for retroperitoneal tumors; experience at Hospital Pablo Tobón Uribe, Medellín, Colombia

Introducción. El abordaje transperitoneal es la técnica más utilizada para la resección de los tumores retroperitoneales. El abordaje extraperitoneal podría ser una alternativa viable para pacientes seleccionados, que no se ha descrito ni evaluado en estudios clínicos. Materiales y métodos. Se llevó a cabo un estudio descriptivo, retrospectivo, de los resultados de dos abordajes para los tumores retroperitoneales: transperitoneal y extraperitoneal. Se revisaron manualmente todas las historias consecutivas de los pacientes con diagnóstico de tumor retroperitoneal, intervenidos quirúrgicamente en el Hospital Pablo Tobón Uribe, desde enero de 2003 hasta marzo de 2011. Resultados. Se evaluaron 37 abordajes, 16 transperitoneales, 19 extraperitoneales y 2 combinados. En 59 % de los pacientes se obtuvo resección R0 (50 % abordaje transperitoneal Vs. 68 % abordaje retroperitoneal). El tiempo quirúrgico promedio fue de 152,96 minutos (159,8 Vs. 134,5). En general, los pacientes no requirieron transfusión de hemoderivados (56,3 Vs. 78,9 %). El 30 % de los paciente presentó complicaciones posoperatorias (25 % Vs. 31,6 %). Hubo una muerte intraoperatoria durante un procedimiento con abordaje extraperitoneal. Discusión. El abordaje extraperitoneal para los tumores retroperitoneales es una técnica nueva. Se requieren más estudios al respecto para aclarar adecuadamente su papel en la resección de los tumores retroperitoneales.

Introduction: The most common technique for resection of retroperitoneal tumors it is by far the transperitoneal approach. The extra peritoneal approach could be a viable alternative for selected patients, but it has not been described nor evaluated in clinical trials. Material and methods: This is a retrospective and descriptive study in which we analyzed the results for the two different approaches for retroperitoneal tumors. The clinical records of all patients with the diagnosis of retroperitoneal tumor that were operated at Hospital Pablo Tobón Uribe during a period of time between January 2003 and March 2011 were reviewed manually. Results: Of a total of 37 surgical procedures, 16 were transperitonal, 19 extraperitoneal and two of them required a combined approach. 59% had a resection graded as R0 (50% with the trans peritoneal approach and 68% with the retroperitoneal approach).The mean operating time was 152.96 minutes (159.8 vs. 134.5, respectively). Most of the patients did not need blood transfusion (56,3 vs 78,9%).The complication rate was 30% (25% vs. 31.6%). One patient that died during an extraperitoneal approach operation. Discussion: The extraperitoneal approach is a new a new technique for the resection of retroperitoneal tumors. More studies are required before a full recommendation favoring this technique can be stated.

Ganglio Postcentinela en Melanoma: nuevo Método Diagnóstico para la detección de Metástasis Ganglionares Pélvicas / Postsentinel lymph node in melanoma: a new diagnostic method for detection for metastatic pelvic lymp nodes

Los ganglios inguinales son el primer sitio de afectación metastásica del melanoma primario de las extremidades inferiores. Cuando se comprueba metástasis ganglionares inguinales no es claro si solamente se debe realizar linfadenectomía inguinal superficial (linfadenectomía ganglionar limitada) o si por el contrario, se debe efectuar linfadenectomía pélvica (cadena ganglionar ilíaca externa y obturador), además de la linfadenectomía inguinal superficial (linfadenectomía ganglionar extendida o combinada). Se propone al ganglio postcentinela como nuevo método diagnóstico capaz de predecir el compromiso ganglionar pélvico y así reducir el número de vaciamientos pélvicos innecesarios.