Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S,and G2 Phases of the Cell Cycle in Trypanosomatids

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU‐ and EdU‐incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and “click” chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re‐estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.     

Long-term Sonographic and Serological Follow-up of Inactive Echinococcal Cysts of the Liver: Hints for a “Watch-and-Wait” Approach

Human cystic echinococcosis is a chronic, complex and neglected infection. Its clinical management has evolved over decades without adequate evaluation of efficacy. Recent expert opinion recommends that uncomplicated inactive cysts of the liver should be left untreated and solely monitored over time (“watch-and-wait” approach). However, clinical data supporting this approach are still scant and published mostly as conference proceedings. In this study, we report our experience with long-term sonographic and serological follow-up of inactive cysts of the liver. From March 1994 to October 2013, 38 patients with 47 liver cysts, diagnosed as inactive without any previous treatment history, were followed with ultrasound and serology at 6–12 months intervals for a period of at least 24 months (median follow-up 51.95 months) in our outpatient clinic. In 97.4% of patients, the cysts remained inactive over time and in only one case was reactivation of the cyst detected. No complications occurred during the time of monitoring. During follow-up, serology tests for CE were negative at diagnosis or became negative in 74.1% and were positive or became positive in 25.9% of cases. Patients with inactive cysts on ultrasound but positive serological tests were also investigated by CT scan (chest and abdomen) to rule out extra-hepatic cyst localization. This study confirms the importance of a stage-specific approach to the management of cystic echinococcosis and supports the use of a monitoring-only approach to inactive, uncomplicated cysts of the liver. It also confirms that serology plays only an ancillary role in the clinical management of these patients, compared to ultrasound and other imaging techniques. The implications of these findings for clinical management and natural history of cystic echinococcosis are discussed.     

Co-composting of chestnut burr and leaf litter with solid poultry manure

A co-composting of chestnut burr and leaf litter mixed with solid poultry manure was assessed by comparison of several chemical, physicochemical and biological parameters. The final pH of the co-compost was 8.89 and the C/N ratio was 13. The germination index (GI) obtained using the co-compost varied with the seeds used. It was 155.35% for ryegrass seeds, 56.56% for wheat seeds and 100% for barley seeds. The co-compost was mature in 103 days from a biological point of view.     

Insulin-like growth factor-binding protein-2 levels in pediatric patients with growth hormone deficiency, eating disorders and acute lymphoblastic leukemia

Insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) is altered in different diseases and might be used as an indication of its severity. The aims of our study were to investigate: (1) the developmental pattern of the serum IGFBP-2 concentration at birth and during childhood and adolescence; (2) whether the serum IGFBP-2 level could be a marker for the diagnosis and evolution of diseases where the growth hormone (GH)-IGF axis is altered, and (3) whether this binding protein shows a relationship with IGF-I, its free fraction, IGFBP-1 and -3. We report reference values for 55 normal full-term newborns and 221 normal children who were divided into 5 groups according to their Tanner stage. Serum levels were higher in newborns when compared with Tanner stages I-V (p < 0.001, ANOVA), with no further changes throughout development. Furthermore, we studied IGFBP-2 levels in 24 children with congenital GH deficiency (GHD), 26 with acute lymphoblastic leukemia (ALL), 75 obese children, and 60 girls with anorexia nervosa (AN) at diagnosis and during a follow-up period. IGFBP-2 at diagnosis was increased in GHD, ALL and AN, and decreased in obesity (p < 0.05, ANOVA). During the follow-up, IGFBP-2 concentrations tended to normalize. IGFBP-2 correlated positively with IGFBP-1 and negatively with IGF-I and IGFBP-3 in normal subjects and at diagnosis of the pathologies studied. Although IGFBP-2 functions are not well understood, these results suggest a possible role for this protein in diseases where the GH-IGF axis is altered.     

Activity of artichoke leaf extract on reactive oxygen species in human leukocytes

Artichoke leaf extract was studied in human leukocytes for activity against oxidative stress using flow cytometry and dichlorofluorescin diacetate as a fluorescence probe. It produces a concentration-dependent inhibition of oxidative stress when cells are stimulated with agents that generate reactive oxygen species (ROS): hydrogen peroxide, phorbol-12-myristate-13-acetate (PMA), and N-formyl-methionyl-leucyl-phenylalanine (FMLP). Cynarin, caffeic acid, chlorogenic acid, and luteolin, constituents of artichoke leaf extract, also show a concentration-dependent inhibitory activity in the above models, contributing to the antioxidant activity of the extract in human neutrophils.     

HmuS and HmuQ of <Emphasis Type="Italic">Ensifer</Emphasis>/<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> degrade heme in vitro and participate in heme metabolism in vivo

Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a K_d value of 5 and 4 µM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-β and IX-δ in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-δ only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo.     

Biosynthesis of poly-β-hydroxybutyrate (PHB) with a high molecular mass by a mutant strain of Azotobacter vinelandii (OPN)

The aim of this study was to characterize the influence of the aeration conditions on the production of PHB and its molecular mass in a mutant strain of Azotobacter vinelandii (OPN), which carries a mutation on ptsN, the gene encoding enzyme IIA^Ntr, previously shown to increase the accumulation of PHB. Cultures of A. vinelandii wild-type strain OP and its mutant derivative strain OPN were grown in 500-mL flasks, containing 100 and 200 mL of PY sucrose medium. PHB production and its molecular mass were analyzed at the end of the culture. The molecular mass (MM) was significantly influenced by the aeration conditions and strain used. A polymer with a higher molecular weight was produced under low aeration conditions for both strains. A maximal molecular mass of 2,026 kDa (equivalent to 3,670 kDa measured by GPC) was obtained with strain OPN cultured under low-aeration conditions, reaching a value two-fold higher than that obtained from the parental strain OP (MM?=?1,013 kDa) grown under the same conditions. Aeration conditions and the ptsN mutation influence the molecular mass of the PHB produced by A. vinelandii affecting in turn its physico-chemical properties.     

Water-Soluble Ruthenium Complexes Bearing Activity Against Protozoan Parasites

Parasitic illnesses are major causes of human disease and misery worldwide. Among them, both amebiasis and Chagas disease, caused by the protozoan parasites, Entamoeba histolytica and Trypanosoma cruzi, are responsible for thousands of annual deaths. The lack of safe and effective chemotherapy and/or the appearance of current drug resistance make the development of novel pharmacological tools for their treatment relevant. In this sense, within the framework of the medicinal inorganic chemistry, metal-based drugs appear to be a good alternative to find a pharmacological answer to parasitic diseases. In this work, novel ruthenium complexes [RuCl₂(HL)(HPTA)₂]Cl₂ with HL = bioactive 5-nitrofuryl containing thiosemicarbazones and PTA?=?1,3,5-triaza-7-phosphaadamantane have been synthesized and fully characterized. PTA was included as co-ligand in order to modulate complexes aqueous solubility. In fact, obtained complexes were water soluble. Their activity against T. cruzi and E. histolytica was evaluated in vitro. [RuCl₂(HL4)(HPTA)₂]Cl₂ complex, with HL4?=?N-phenyl-5-nitrofuryl-thiosemicarbazone, was the most active compound against both parasites. In particular, it showed an excellent activity against E. histolytica (half maximal inhibitory concentration (IC₅₀)?=?5.2 μM), even higher than that of the reference drug metronidazole. In addition, this complex turns out to be selective for E. histolytica (selectivity index (SI) >38). The potential mechanism of antiparasitic action of the obtained ruthenium complexes could involve oxidative stress for both parasites. Additionally, complexes could interact with DNA as second potential target by an intercalative-like mode. Obtained results could be considered a contribution in the search for metal compounds that could be active against multiple parasites.