Kinetic and Structural Studies of Aldehyde Oxidoreductase from Desulfovibrio gigas Reveal a Dithiolene-Based Chemistry for Enzyme Activation and Inhibition by H2O2

Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) family catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum active site shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. The XO family member aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is an exception as presents in its catalytically competent form an equatorial oxo ligand instead of the sulfido ligand. Despite this structural difference, inactive samples of DgAOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that DgAOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive DgAOR samples is different to that of desulfo-XO. We now report a combined kinetic and X-ray crystallographic study to unveil the enzyme modification responsible for the inactivation and the chemistry that occurs at the Mo site when DgAOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, DgAOR activation/inactivation is governed by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent behavior of the pyranopterin in enzyme activity. We also showed that DgAOR incubation with dithionite plus sulfide in the presence of dioxygen produces hydrogen peroxide not associated with the enzyme activation. The peroxide molecule coordinates to molybdenum in a η² fashion inhibiting the enzyme activity.     

Metacercariae of Galactosomum lacteum (Jägerskiöld, 1896) Looss, 1899 (Heterophyidae) from marine teleosts in the Gulf of Cagliari (southern Sardinia, Italy)

Galactosomum lacteum (J?gerski?ld, 1896) Looss, 1899 metacercariae, encysted on the optic nerve, on the brain and/or on the muscle and the connective of the pharynx and oesophagus, were found in Spicara maena L., S. flexuosa Rafinesque, 1810, S. smaris L. (Centracanthidae), Gobius cruentatus Gmelin, 1789 (Gobiidae), Symphodus tinca L., S. mediterraneus L. (Labridae), Serranus cabrilla L. (Serranidae), Diplodus sargus L. and D. annularis L. (Sparidae) caught in the Gulf of Cagliari (southern Sardinia, Italy). Excysted specimens were identified by some distinctive morphological features: more or less expanded forebody, depending on whether the specimens were living or fixed; tubular excretory bladder extending to the posterior border of the ovary; two-chambered seminal vesicle; asymmetrical and parenchymatous ventral sucker with lines of spines within its cavity; and unarmed gonotyle. Comparison has been made with the congeneric species metacercaria, G. timondavidi Pearson & Prévot, 1971, also registered in the Mediterranean Sea.     

Athletes’ rest-activity circadian rhythm differs in accordance with the sport discipline

The correct expression of circadian rhythmicity is crucial for the body homeostasis. The rest-activity circadian rhythms (RARs) are involved in the control of the sleep-wake cycle and altered RARs could lead to a compromised health status. Many studies focused on examining sleep behavior and circadian rhythms in physically active subjects or athletes but, unexpectedly, no data on RARs are available. Therefore, we studied the existence of the RAR in athletes and the possible difference in RAR’s characteristics among sport disciplines. The study had a prospective observational design and RARs were recorded for five consecutive training days through actigraphy (Actiwatch 2 actigraph; Philips Respironics, OR, USA) in 43 athletes (mean age: 25.6 ± 3.2 years). Athletes competed in three different disciplines and had different training schedules and competition levels: professional triathletes (N = 10; 6 females and 4 males) had 2 morning (08:30–12:00) and 1 afternoon (15:00–17:00) training sessions, professional volleyball players (N = 19; 12 females and 7 males) used to train once in the morning (09:00–11:30) and once in the afternoon (15:00–18:00), and non-professional soccer players (N = 14; all males) trained always late in the evening (20:30–22:30). To determine the existence of RARs, the activity counts (A.C.) data were analyzed using the single and the population mean cosinor method; a one-way analysis of variance (ANOVA) followed by the Tukey–Kramer post-hoc test was used for the comparison of RAR characteristics among soccer, volleyball and triathlon athletes. Partial eta squared (?_p²) was used to determine the magnitude of the effect for significant outcomes (α = 0.05) in ANOVA. The presence of a significant RAR both for each of the 43 athletes (p < 0.001) and for the three categories of athletes (p < 0.001) was observed. RARs differed among sport disciplines: the Midline Estimating Statistic of Rhythm (MESOR) was significantly higher in triathletes (mean: 347 A.C. with 95% Confidence Interval [CI]: 314–379) compared to both volleyball (mean: 188 A.C. with 95% CI: 173–203; p < 0.001) and soccer players (mean: 289 A.C. with 95% CI: 267–312; p < 0.01) with ?_p² = 0.72. Amplitude (A) values showed the same significant trend of MESOR data (ANOVA: p < 0.001; ?_p² = 0.65) while the acrophase (Φ) occurred at 18:28 for soccer players, significantly later than triathlon (15:20 h; p < 0.001) and volleyball players (16:24 h; p < 0.001) (ANOVA: p < 0.001; ?p2 = 0.84). The higher training duration and intensity reached by triathlon athletes in the morning sessions caused a phase advance of their RAR’s acrophase Φ and higher MESOR and A amplitude compared to volleyball players and triathletes. Therefore, different sport disciplines require different training schedules, training loads and intensities that translate into different RARs. Strength coaches and medical staff of professional teams should strongly consider actigraphy as a practical and powerful tool to monitor RARs, sleep behavior, and the activity levels of their athletes; highlighting potential circadian disruptions through actigraphy could be helpful to prevent musculoskeletal injuries.     

Microglia of medicinal leech (Hirudo medicinalis) express a specific activation marker homologous to vertebrate ionized calcium‐binding adapter molecule 1 (Iba1/alias aif‐1)

The Ionized calcium‐Binding Adapter molecule 1 (Iba1), also known as Allograft Inflammatory Factor 1 (AIF‐1), is a 17 kDa cytokine‐inducible protein, produced by activated macrophages during chronic transplant rejection and inflammatory reactions in Vertebrates. In mammalian central nervous system (CNS), Iba1 is a sensitive marker associated with activated macrophages/microglia and is upregulated following neuronal death or brain lesions. The medicinal leech Hirudo medicinalis is able to regenerate its CNS after injury, leading to a complete functional repair. Similar to Vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. We identified a gene, named Hmiba1, coding a 17.8 kDa protein showing high similarity with Vertebrate AIF‐1. The present work constitutes the first report on an Iba1 protein in the nervous system of an invertebrate. Immunochemistry and gene expression analyses showed that HmIba1, like its mammalian counterpart, is modulated in leech CNS by mechanical injury or chemical stimuli (ATP). We presently demonstrate that most of leech microglial cells migrating and accumulating at the lesion site specifically expressed the activation marker HmIba1. While the functional role of Iba1, whatever species, is still unclear in reactive microglia, this molecule appeared as a good selective marker of activated cells in leech and presents an interesting tool to investigate the functions of these cells during nerve repair events. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 987–1001, 2014     

Increasing Strawberry Fruit Sensorial and Nutritional Quality Using Wild and Cultivated Germplasm

Background

Increasing antioxidant levels in fruit through breeding is an important option to support higher antioxidant intake particularly when fruit consumption is low. Indeed, if nutritional components are also combined with a high standard of sensorial fruit quality, the perspective for consumer health can be further improved by encouraging more fruit consumption. Wild species are valued by strawberry breeders as sources of novel traits, especially for pest resistance and abiotic stress tolerance. Furthermore, previous investigations have shown improvements in fruit nutritional quality in breeding material that originated from Fragaria virginiana ssp. glauca (FVG) inter-species crosses. Recently, commercial varieties of strawberries have also shown interesting variability in fruit nutritional quality.

Results

Strawberry fruit sensorial and nutritional qualities generated by Fragaria inter-species and intra-species crosses were evaluated on 78 offspring derived from 8 families: two that originated from F. × ananassa intra-species crossing; three from back-crossing of F1– FVG × F. × ananassa; and three from back-crossing of BC1– FVG × F. × ananassa. The genetic variability from the three types of cross combinations was analyzed by calculation of the correlations among the fruit sensorial and nutritional parameters. The results obtained show that two subsequent back-crossing generations from an inter-species crossing combination with F. virginiana ssp. glauca provides useful improvement of the fruit nutritional and sensorial qualities that is combined with agronomic standards that are close to those requested at the commercial level. Improvements of these traits can also be achieved by programming F. × ananassa intra-species crosses and producing progeny with productivity traits more similar to those of the commercial cultivars.

Conclusions

The two types of combination programs (inter-species back-crosses, and intra-species crosses) can be used to improve strawberry nutritional quality.     

Spontaneous splenic rupture in patient with metastatic melanoma treated with vemurafenib

ABSTRACT: BACKGROUND: BRAF inhibitors such as vemurafenib are a new family of biological drugs, recently available to treat metastatic malignant melanoma. METHODS: We present the case of a 38-year-old man affected by metastatic melanoma who had been under treatment with vemurafenib for a few days. The patient suffered from sudden onset of abdominal pain due to intra-abdominal hemorrhage with profuse hemoperitoneum. An emergency abdominal sonography confirmed the clinical suspicion of a splenic rupture. RESULTS: The intraoperative finding was hemoperitoneum due to splenic two-step rupture and splenectomy was therefore performed. Histopathology confirmed splenic hematoma and capsule laceration, in the absence of metastasis. CONCLUSIONS: This report describes the occurrence of a previously unreported adverse event in a patient with stage IV melanoma receiving vemurafenib.     

A metabolomics-based approach for non-invasive diagnosis of chromosomal anomalies

Introduction

Chromosomal anomalies (CA) are the most frequent fetal anomalies.

Objective

To evaluate the diagnostic performance of a machine learning ensemble model based on the maternal serum metabolomic fingerprint of fetal aneuploidies during the second trimester .

Methods

This is a case-control pilot study. Metabolomic profiles have been obtained on serum of 328 mothers (220 controls and 108 cases), using gas chromatography coupled to mass spectrometry. Eight machines learning and classification models were built and optimized. An ensemble model was built using a voting scheme. All samples were randomly divided into two sets. One was used as training set, the other one for diagnostic performance assessment.

Results

Ensemble machine learning model correctly classified all cases and controls. The accuracy was the same for trisomy 21 and 18; also, the other CA were correctly detected. Elaidic, stearic, linolenic, myristic, benzoic, citric and glyceric acid, mannose, 2-hydroxy butyrate, phenylalanine, proline, alanine and 3-methyl histidine were selected as the most relevant metabolites in class separation.

Conclusion

The proposed model, based on the maternal serum metabolomic fingerprint of fetal aneuploidies during the second trimester, correctly identifies all the cases of chromosomal abnormalities. Overall, this preliminary analysis appeared suggestive of a metabolic environment conductive to increased oxidative stress and a disturbance in the fetal central nervous system development. Maternal serum metabolomics can be a promising tool in the screening of chromosomal defects. Moreover, metabolomics allows to extend our knowledge about biochemical alterations caused by aneuploidies and responsible for the observed phenotypes.

     

Salivary cortisol concentration after high-intensity interval exercise: Time of day and chronotype effect

Due to personal and working necessities, the time for exercise is often short, and scheduled early in the morning or late in the afternoon. Cortisol plays a central role in the physiological and behavioral response to a physical challenge and can be considered as an index of exercise stress. Therefore, the aim of this study was to evaluate the influence of the circadian phenotype classification on salivary cortisol concentration in relation to an acute session of high-intensity interval exercise (HIIE) performed at different times of the day. Based on the morningness–eveningness questionnaire, 12 M-types (N = 12; age 21 ± 2 years; height 179 ± 5 cm; body mass 74 ± 12 kg, weekly training volume 8 ± 1 hours) and 11 E-types (N = 11; age 21 ± 2 years; height 181 ± 11 cm; body mass 76 ± 11 kg, weekly training volume 7 ± 2 hours) were enrolled in a randomized crossover study. All subjects underwent measurements of salivary cortisol secretion before (PRE), immediately after (POST), and 15 min (+15 min), 30 min (+30 min), 45 min (+45 min) and 60 min (+60 min) after the completion of both morning (08.00 am) and evening (08.00 p.m.) high-intensity interval exercise. Two-way analysis of variance with Tuckey’s multiple comparisons test showed significant increments over PRE-cortisol concentrations in POSTcondition both in the morning (4.88 ± 1.19 ng · mL^?1 vs 6.60 ± 1.86 ng · mL^?1, +26.1%, P < 0.0001, d > 0.8) and in the evening (1.56 ± 0.48 ng · mL^?1 vs 2.34 ± 0.37, +33.4%, P = 0.034, d > 0.6) exercise in all the 23 subject that performed the morning and the evening HIIE. In addition, during morning exercise, significant differences in cortisol concentration between M-types and E-types at POST (5.49 ± 0.98 ng · mL^?1 versus 8.44 ± 1.08 ng · mL^?1, +35%, P < 0.0001, d > 0.8), +15 min (4.52 ± 0.42 ng · mL^?1 versus 6.61 ± 0.62 ng · mL^?1, +31.6%, P < 0.0001, d > 0.8), +30 min (4.10 ± 1.44 ng · mL^?1 versus 6.21 ± 1.60 ng · mL^?1, +34.0%, P < 0.0001, d = 0.7), + 45 min (3.78 ± 0.55 ng · mL^?1 versus 5.80 ± 0.72 ng · mL^?1, +34.9%, P < 0.0001, d = 0.7), and + 60 min condition(3.53 ± 0.45 ng · mL^?1 versus 5.78 ± 1.13 ng · mL^?1, 38.9%, P = 0.0008, d = 0.7) were noted. No statistical significant differences between M-types and E-types during evening HIIE on post-exercise cortisol concentration were detected. E-types showed a higher morning peak of salivary cortisol respect to M-types when performing a HIIE early in the morning and produced higher salivary cortisol concentrations after the cessation of the exercise. Practical applications suggest that it is increasingly important for the exercise professionals to identify the compatibility between time of day for exercising and chronotype to find the individual’s favorable circadian time to perform a HIIE.     

Orchis patens Desf.: seed morphology of an endangered Mediterranean orchid

The loss of orchid habitats has increased the investment on orchid conservation efforts to reduce the risk of extinction of rare species. In northwestern Italy, orchids are among the most threatened of all plant groups in the region, but little is known about the biology of most of the less abundant taxa. In this study, we used light and scanning electron microscopy to characterize the morphology of the seeds of the threatened Orchis patens Desf., a declining species present in semi-natural habitats. We found a marked positive relationship between seed size and embryo size that may be not restricted to O. patens but also occur in other orchid species. The comparative analysis of the observed seed traits revealed hidden morphological affinities at the reproductive level among O. patens and the three subspecies of O. mascula, showing the potential of this approach to test the taxonomic relationships among the different taxa included in the genus Orchis.