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.     

Physical performance in kidney transplanted patients: a study on desert trekking

Physical performance of kidney transplanted patients in challenging environments, such as deserts, has been poorly studied. Six kidney transplanted (T: 5 males, 1 female; 45±6 yrs) and 8 control (C: 5 males, 3 females; 49±13 yrs) subjects participated in a 5-day desert trek. Blood pressure, hydration status (Height2/Rz by bioimpedance), heart rate, energy expenditure (by SenseWear Pro Armband) and walking velocities were recorded during each daily trekking stage (GPS-assisted wearable devices). Systo-diastolic blood pressure did not differ between C (119/77±12/8 mmHg) and T (121/77±10/6 mmHg) groups throughout the study. The hydration status was stable from day 1 (Ht2/Rz: 64±13 cm2/Ohm in T and 59±12 cm2/Ohm in C subjects) to day 5 (66±11 cm2/Ohm in T and 61±13 cm2/Ohm in C subjects) in both groups. Two patients on steroid treatment showed a relative hyperhydration. Mean heart rate did not differ between T (135±10 bpm) and C (136±5 bpm) subjects throughout the study, although a reduction from day 1 to day 5 was observed in T subjects only (p<0.05 vs C group). No differences were found between T and C group in walking velocity (1.7±0.6 km/h in T and 1.7±0.5 km/h in C group); mean intensity of physical activity was 3.4±0.5 METs in T and 3.3±0.6 METs in C group during each trekking stage. Negligible differences were observed in cardiovascular, metabolic and hydration status adaptations to desert trekking between selected T and C individuals. T subjects with creatinine clearance > 55 ml/min showed acceptable physical performance and acclimatization to desert environment, suggesting a good long-term outcome of transplantation.     

Chestnut cultivar diversification process in the Iberian Peninsula, Canary Islands, and Azores

This is a large-scale molecular study based on simple sequence repeat (SSR) loci of the diversification process in chestnut cultivars from Portugal and Spain, from the northern Iberian Peninsula to the Canary Islands and the Azores. A total of 593 grafted chestnut trees (Castanea sativa Mill.) were analysed with 10 SSRs: 292 from Portugal and 301 from Spain. Some of the trees studied were more than 300 years old. Accessions were analysed using a model-based Bayesian procedure to assess the geographical structure and to assign individuals to reconstructed populations based on the SSR genotypes. We found 356 different genotypes with a mean value of clonality of 33% owing to grafting. Mutations accounted for 6%, with hybridization being the main diversification process that can explain the great diversity found. Ten main cultivar groups were detected: four in northern Spain, five in the centre of the Iberian Peninsula, and one in southern Spain related to the centre of the Iberian Peninsula. This work demonstrated that cultivar origin and the diversification process was a combination of clonal propagation of selected seedlings, hybridization, and mutations, which allowed high levels of diversity to be maintained with respect to selected clones for fruit production. Furthermore, seedlings and graft sticks facilitated the transport to new destinations in the colonization process, transporting sometimes more than 3000 km if we consider the Azores and the Canary Islands.     

Phytochemical Characterization of Cannabis sativa L. Roots from Northeastern Brazil

This article describes the phytochemical study of Cannabis sativa roots from northeastern Brazil. The dried plant material was pulverized and subjected to exhaustive maceration with ethanol at room temperature, obtaining the crude ethanolic extract (Cs-EEBR). The volatile compounds were analyzed by gas chromatography coupled with mass spectrometry (GC/MS), which allowed to identify 22 compounds by comparing the linear retention index (LRI), the similarity index (SI) and the fragmentation pattern of the constituents with the literature. By this technique the major compounds identified were: friedelan-3-one and β-sitosterol. In addition, two fractions were obtained from Cs-EEBR by classical column chromatography and preparative thin layer chromatography. These fractions were analyzed by NMR and IR and together with the mass spectrometry data allowed to identify the compounds: epifriedelanol, friedelan-3-one, β-sitosterol and stigmasterol. The study contributed to the phytochemical knowledge of Cannabis sativa, specifically the roots, as there are few reports on the chemical constituents of this part of the plant.     

Sugar metabolism and developmental stages of rubber tree (Hevea brasiliensis L.) seeds

Changes in the concentration of sugars and sucrose metabolism enzymes can characterize the developmental stages of a seed. In recalcitrant species such as Hevea brasiliensis L., little is known about these changes. We aimed to evaluate the three main stages of development of rubber tree seeds – histodifferentiation, cell elongation and accumulation of reserves. The activities of acid and neutral invertases (E.C. 3.2.1.26) and sucrose synthase (EC 2.4.1.13), and the concentrations of reducing sugars (RS), total soluble sugars (TSS) and sucrose (Suc) were determined concomitantly with the histochemical and anatomical evaluation of seed structure. Histodifferentiation in rubber tree seeds occurs up to 75 days after anthesis (DAA). The concentration of RS is high and of Suc is low during seed histodifferentiation, which occurs along with a visible increase in the number of cell divisions. After that period, there is an increase in the concentration of Suc (mg g^?1) and in the number and size of starch granules, and a decrease in the concentration of RS (mg g^?1). At that point, cell elongation occurs. At 135 DAA, there is an inversion in the concentration of these two sugars and an increase in reserve accumulation. Thus, in seeds of the evaluated clone, the period up to 75 DAA is characterized as the histodifferentiation stage, while from that time up to 120 DAA the cell elongation stage takes place. The final stage of seed maturation and reserve accumulation begins at 135 DAA, and the seed, including the embryo, is completely formed at 175 DAA.     

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

Applied Microbiology and Biotechnology - Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate...     

The eukaryotic nucleotide excision repair pathway

Nucleotide excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PPs). In this review, we describe the main protein players and the different sequential steps of the eukaryotic NER mechanism in human cells, from lesion recognition to damage removal and DNA synthesis. Studies on the dynamics of protein access to the damaged site, and the kinetics of lesion removal contribute to the knowledge of how the cells respond to genetic insult. DNA lesions as well as NER factors themselves are also implicated in changes in cell metabolism, influencing cell cycle progression or arrest, apoptosis and genetic instability. These changes are related to increased mutagenesis and carcinogenesis. Finally, the recent collection of genomic data allows one to recognize the high conservation and the evolution of eukaryotic NER. The distribution of NER orthologues in different organisms, from archaea to the metazoa, displays challenging observations. Some of NER proteins are widespread in nature, probably representing ancient DNA repair proteins, which are candidates to participate in a primitive NER mechanism.     

MicroRNAs: new players in heart failure

MicroRNAs (miRNAs) are a class of non-coding small RNAs representing one of the most exciting areas of modern medical science. miRNAs modulate a large and complex regulatory network of gene expression of the majority of the protein-coding genes. Currently, evidences suggest that miRNAs play a crucial role in the pathogenesis of heart failure. Some miRNAs as miR-1, miR-133 and miR-208a are highly expressed in the heart and strongly associated with the development of cardiac hypertrophy. Recent data indicate that these miRNAs as well as miR-206 change their expression quickly in response to physical activity. The differential regulation of miRNAs in response to exercise suggests a potential value of circulating miRNAs (c-miRNAs) as biomarkers of physiological mediators of the cardiovascular adaptation induced by exercise. Likewise, serum levels of c-miRNAs such as miR-423-5p have been evaluated as potential biomarkers in the diagnosis and prognosis of heart failure. On the other hand, the manipulation of miRNAs levels using techniques such as ‘miR mimics’ and ‘antagomiRs’ is becoming evident the enormous potential of miRNAs as promising therapeutic strategies in heart failure.     

Effects of Anabolic Steroids and High-Intensity Aerobic Exercise on Skeletal Muscle of Transgenic Mice

In an attempt to shorten recovery time and improve performance, strength and endurance athletes occasionally turn to the illicit use of anabolic-androgenic steroids (AAS). This study evaluated the effects of AAS treatment on the muscle mass and phenotypic characteristics of transgenic mice subjected to a high-intensity, aerobic training program (5d/wk for 6 weeks). The transgenic mice (CETP^+/-LDLr^-/+) were engineered to exhibit a lipid profile closer to humans. Animals were divided into groups of sedentary (Sed) and/or training (Ex) mice (each treated orally with AAS or gum arabic/vehicle: Sed-C, Sed-M, ex-C, ex-M). The effects of AAS (mesterolone: M) on specific phenotypic adaptations (muscle wet weight, cross-sectional area, and fiber type composition) in three hindlimb muscles (soleus:SOL, tibialis anterior:TA and gastrocnemius:GAS) were assessed. In order to detect subtle changes in fiber type profile, the entire range of fiber types (I, IC, IIAC, IIA, IIAD, IID, IIDB, IIB) was delineated using mATPase histochemistry. Body weight gain occurred throughout the study for all groups. However, the body weight gain was significantly minimized with exercise. This effect was blunted with mesterolone treatment. Both AAS treatment (Sed-M) and high-intensity, aerobic training (ex-C) increased the wet weights of all three muscles and induced differential hypertrophy of pure and hybrid fibers. Combination of AAS and training (ex-M) resulted in enhanced hypertrophy. In the SOL, mesterolone treatment (Sed-M and ex-M) caused dramatic increases in the percentages of fiber types IC, IIAC, IIAD, IID, with concomitant decrease in IIA, but had minimal impact on fiber type percentages in the predominantly fast muscles. Overall, the AAS-induced differential adaptive changes amounted to significant fiber type transformations in the fast-to-slow direction in SOL. AAS treatment had a significant effect on muscle weights and fiber type composition in SOL, TA and GAS which was even maximized in animals subjected to metabolically high-intensity aerobic exercise.     

Trypanosoma cruzi: cloning and expression of an antigen recognized by acute and chronic human chagasic sera.

Here we describe the characterization of a Trypanosoma cruzi DNA sequence (clone A13) that codes for a polypeptide recognized by IgM and IgG antibodies from sera of acute and congenital chagasic patients. Antibodies to A13 antigen are also detected in the sera of chronic patients with different clinical forms of Chagas' disease, but not in sera of patients with leishmaniasis or other parasitic diseases. The antigenic determinants encoded by clone A13 are found in amastigotes and trypomastigotes of several T. cruzi strains, but not in the noninfective epimastigotes. The DNA sequence of the recombinant clone reveals one open reading frame encoding 251 amino acids without tandemly repeated sequences. Our data suggest that the A13 antigen may be useful for the development of serodiagnostic procedures.