SUMMARY The selective inhibition of bacterial β-glucuronidases was recently shown to alleviate drug-induced gastrointestinal toxicity in mice, including the damage caused by the widely used anticancer drug irinotecan. Here, we report crystal structures of representative β-glucuronidases from the Firmicutes Streptococcus agalactiae and Clostridium perfringens and the Proteobacterium Escherichia coli, and the characterization of a β-glucuronidase from the Bacteroidetes Bacteroides fragilis. While largely similar in structure, these enzymes exhibit marked differences in catalytic properties and propensities for inhibition, indicating that the microbiome maintains functional diversity in orthologous enzymes. Small changes in the structure of designed inhibitors can induce significant conformational changes in the β-glucuronidase active site. Finally, we establish that β-glucuronidase inhibition does not alter the serum pharmacokinetics of irinotecan or its metabolites in mice. Together, the data presented advance our in vitro and in vivo understanding of the microbial β-glucuronidases, a promising new set of targets for controlling drug-induced gastrointestinal toxicity.

In humans, intrauterine growth restriction (IUGR) is associated with vascular dysfunction, oxidative stress and signs of endothelial programming in umbilical vessels. We aimed to determine the effects of maternal antioxidant treatment with N-acetylcysteine (NAC) on fetal endothelial function and endothelial nitric oxide synthase (eNOS) programming in IUGR guinea pigs. IUGR was induced by implanting ameroid constrictors on uterine arteries of pregnant guinea pigs at mid gestation, half of the sows receiving NAC in the drinking water (from day 34 until term). Fetal biometry and placental vascular resistance were followed by ultrasound throughout gestation. At term, umbilical arteries and fetal aortae were isolated to assess endothelial function by wire-myography. Primary cultures of endothelial cells (ECs) from fetal aorta, femoral and umbilical arteries were used to determine eNOS mRNA levels by quantitative PCR and analyse DNA methylation in the Nos3 promoter by pyrosequencing. Doppler ultrasound measurements showed that NAC reduced placental vascular resistance in IUGR (P < 0.05) and recovered fetal weight (P < 0.05), increasing fetal-to-placental ratio at term (∼40%) (P < 0.001). In IUGR, NAC treatment restored eNOS-dependent relaxation in aorta and umbilical arteries (P < 0.05), normalizing eNOS mRNA levels in EC fetal and umbilical arteries (P < 0.05). IUGR-derived ECs had a decreased DNA methylation (∼30%) at CpG -170 (from the transcription start site) and this epigenetic signature was absent in NAC-treated fetuses (P < 0.001). These data show that IUGR-ECs have common molecular markers of eNOS programming in umbilical and systemic arteries and this effect is prevented by maternal treatment with antioxidants.

Background: Chronic hypoxia and oxidative stress during gestation lead to pulmonary hypertension of the neonate (PHN), a condition characterized by abnormal pulmonary arterial reactivity and remodeling. Melatonin has strong antioxidant properties and improves pulmonary vascular function. Here, we aimed to study the effects of melatonin on the function and structure of pulmonary arteries from PHN lambs.Methods: Twelve lambs (Ovis aries) gestated and born at highlands (3,600 m) were instrumented with systemic and pulmonary catheters. Six of them were assigned to the control group (CN, oral vehicle) and 6 were treated with melatonin (MN, 1 mg.kg−1.d−1) during 10 days. At the end of treatment, we performed a graded oxygenation protocol to assess cardiopulmonary responses to inspired oxygen variations. Further, we obtained lung and pulmonary trunk samples for histology, molecular biology, and immunohistochemistry determinations.Results: Melatonin reduced the in vivo pulmonary pressor response to oxygenation changes. In addition, melatonin decreased cellular density of the media and diminished the proliferation marker KI67 in resistance vessels and pulmonary trunk (p < 0.05). This was associated with a decreased in the remodeling markers α-actin (CN 1.28 ± 0.18 vs. MN 0.77 ± 0.04, p < 0.05) and smoothelin-B (CN 2.13 ± 0.31 vs. MN 0.88 ± 0.27, p < 0.05). Further, melatonin increased vascular density by 134% and vascular luminal surface by 173% (p < 0.05). Finally, melatonin decreased nitrotyrosine, an oxidative stress marker, in small pulmonary vessels (CN 5.12 ± 0.84 vs. MN 1.14 ± 0.34, p < 0.05).Conclusion: Postnatal administration of melatonin blunts the cardiopulmonary response to hypoxia, reduces the pathological vascular remodeling, and increases angiogenesis in pulmonary hypertensive neonatal lambs.These effects improve the pulmonary vascular structure and function in the neonatal period under chronic hypoxia.

Pulmonary arterial hypertension of the neonate (PAHN) is a pathophysiological condition characterized by maladaptive pulmonary vascular remodeling and abnormal contractile reactivity. This is a multifactorial syndrome with chronic hypoxia and oxidative stress as main etiological drivers, and with limited effectiveness in therapeutic approaches. Melatonin is a neurohormone with antioxidant and vasodilator properties at the pulmonary level. Therefore, this study aims to test whether a postnatal treatment with melatonin during the neonatal period improves in a long-lasting manner the clinical condition of PAHN. Ten newborn lambs gestated and born at 3600 m were used in this study, five received vehicle and five received melatonin in daily doses of 1 mg kg −1 for the first 3 weeks of life. After 1 week of treatment completion, lung tissue and small pulmonary arteries (SPA) were collected for wire myography, molecular biology, and morphostructural analyses. Melatonin decreased pulmonary arterial pressure the first 4 days of treatment. At 1 month old, melatonin decreased the contractile response to the vasoconstrictors K + , TX 2 , and ET-1. Further, melatonin increased the endothelium-dependent and muscle-dependent vasodilation of SPA. Finally, the treatment decreased pulmonary oxidative stress by inducing antioxidant enzymes and diminishing pro-oxidant sources. In conclusion, melatonin improved vascular reactivity and oxidative stress at the pulmonary level in PAHN lambs gestated and born in chronic hypoxia. K E Y W O R D Schronic hypoxia, melatonin treatment, oxidative stress, pulmonary hypertension of the neonate, reactive oxygen species

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Pulmonary arterial hypertension of newborns (PAHN) constitutes a critical condition involving both severe cardiac remodeling and right ventricle dysfunction. One main cause of this condition is perinatal hypoxia and oxidative stress. Thus, it is a public health concern for populations living above 2500 m and in cases of intrauterine chronic hypoxia in lowlands. Still, pulmonary and cardiac impairments in PAHN lack effective treatments. Previously we have shown the beneficial effects of neonatal melatonin treatment on pulmonary circulation. However, the cardiac effects of this treatment are unknown. In this study, we assessed whether melatonin improves cardiac function and modulates right ventricle (RV) oxidative stress. Ten lambs were gestated, born, and raised at 3600 m. Lambs were divided in two groups. One received daily vehicle as control, and another received daily melatonin (1 mg·kg−1·d−1) for 21 days. Daily cardiovascular measurements were recorded and, at 29 days old, cardiac tissue was collected. Melatonin decreased pulmonary arterial pressure at the end of the experimental period. In addition, melatonin enhanced manganese superoxide dismutase and catalase (CAT) expression, while increasing CAT activity in RV. This was associated with a decrease in superoxide anion generation at the mitochondria and NADPH oxidases in RV. Finally, these effects were associated with a marked decrease of oxidative stress markers in RV. These findings support the cardioprotective effects of an oral administration of melatonin in newborns that suffer from developmental chronic hypoxia.

Stroke is the second leading cause of death worldwide, estimated that 1/6 of the world population will suffer it once in their life. The most common type of this medical condition is the ischemic stroke (IS), produced by a thrombotic or embolic occlusion of a major cerebral artery or its branches, leading to the formation of a complex infarct region caused by oxidative stress, excitotoxicity and endothelial dysfunction. Nowadays, the immediate treatment for IS involves thrombolytic agents or mechanical thrombectomy, depending on the integrity of the blood-brain barrier (BBB). A common stroke complication is the hemorrhagic transformation (HT), which consists of bleeding into the ischemic brain area. Currently, better treatments for IS are urgently needed. As such, the neurohormone melatonin has been proposed as a good candidate due to its antioxidant, anti-inflammatory and neuroprotective effects, particularly against lipid peroxidation and oxidative stress during brain ischemia. Here, we proposed to develop intravenous or intranasal melatonin nanoformulation to specifically target the brain in stroke patients. Nowadays, the challenge is to find a formulation able to cross the barriers and reach the target organ in an effective dose to generate the pharmacological effect. In this review, we discuss the current literature about stroke pathophysiology, melatonin properties and its potential use in nanoformulations as a novel therapeutic approach for ischemic stroke.

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