Study of propidium iodide binding to DNA in intact cells by flow cytometry.

We studied the in situ binding of propidium iodide to DNA in fixed human lymphocytes, using flow cytometry. Experimental data of fluorescence emission vs dye concentration and vs cell concentration were obtained. Data were interpreted by means of two different mathematical models specific for the staining reaction, and the binding parameters were obtained by "best-fitting" of the data. A model based on two classes of binding sites with different affinity constants gave the most satisfactory fitting. The accessibility of the in situ chromatin turned out to be reduced with respect to the non in situ accessibility for ethidium bromide as reported in the literature. The present study shows the usefulness of the flow-cytometric technique for probing DNA structure in intact cells.     

Fish biodiversity of Saint Peter and Saint Paulʼs Archipelago,Mid-Atlantic Ridge,Brazil: new records and a species database

Saint Peter and Saint Paul's Archipelago (SPSPA), one of the smallest and most isolated island groups in the world, is situated on the Mid-Atlantic Ridge, between Brazil and the African continent. SPSPA has low species richness and high endemism; nonetheless, the diversity of fishes from deep habitats (>30 m depth) had not been previously studied in detail. Several expeditions conducted between 2009 and 2018 explored the shallow and deep reefs of SPSPA using scuba, closed-circuit rebreathers, manned submersibles, baited remote underwater stereo-videos (stereo-BRUV) and fishing between 0 and 1050 m depth. These expeditions yielded 41 new records of fishes for SPSPA: 9 in open waters, 9 in shallow waters (0–30 m), 8 in mesophotic ecosystems (30–150 m) and 15 in deeper reefs (>150 m). Combined with literature records of adult pelagic, shallow and deep-reef species, as well as larvae, the database of the fish biodiversity for SPSPA currently comprises 225 species (169 recorded as adult fishes and 79 as larvae, with 23 species found in both stages). Most of them (112) are pelagic, 86 are reef-associated species and 27 are deep-water specialists. Species accumulation curves show that the number of fish species has not yet reached an asymptote. Whereas the number of species recorded in SPSPA is similar to that in other oceanic islands in the Atlantic Ocean, the proportion of shorefishes is relatively lower, and the endemism level is the third highest in the Atlantic. Twenty-nine species are listed as threatened with extinction. Observations confirm the paucity of top predators on shallow rocky reefs of the island, despite the presence of several pelagic shark species around SPSPA. Because all of the endemic species are reef associated, it is argued that the new marine-protected areas created by the Brazilian government do not ensure the protection and recovery of SPSPA's biodiversity because they allow exploitation of the most vulnerable species around the archipelago itself. This study suggests a ban on reef fish exploitation inside an area delimited by the 1000 m isobath around the islands (where all known endemics are concentrated) as the main conservation strategy to be included in the SPSPA management plan being prepared by the Brazilian government.     

GEDAE-LaB: A Free Software to Calculate the Energy System Contributions during Exercise

Purpose

The aim of the current study is to describe the functionality of free software developed for energy system contributions and energy expenditure calculation during exercise, namely GEDAE-LaB.

Methods

Eleven participants performed the following tests: 1) a maximal cycling incremental test to measure the ventilatory threshold and maximal oxygen uptake (V˙O₂max); 2) a cycling workload constant test at moderate domain (90% ventilatory threshold); 3) a cycling workload constant test at severe domain (110% V˙O₂max). Oxygen uptake and plasma lactate were measured during the tests. The contributions of the aerobic (A_MET), anaerobic lactic (LA_MET), and anaerobic alactic (AL_MET) systems were calculated based on the oxygen uptake during exercise, the oxygen energy equivalents provided by lactate accumulation, and the fast component of excess post-exercise oxygen consumption, respectively. In order to assess the intra-investigator variation, four different investigators performed the analyses independently using GEDAE-LaB. A direct comparison with commercial software was also provided.

Results

All subjects completed 10 min of exercise at moderate domain, while the time to exhaustion at severe domain was 144 ± 65 s. The A_MET, LA_MET, and AL_MET contributions during moderate domain were about 93, 2, and 5%, respectively. The A_MET, LA_MET, and AL_MET contributions during severe domain were about 66, 21, and 13%, respectively. No statistical differences were found between the energy system contributions and energy expenditure obtained by GEDAE-LaB and commercial software for both moderate and severe domains (P > 0.05). The ICC revealed that these estimates were highly reliable among the four investigators for both moderate and severe domains (all ICC ≥ 0.94).

Conclusion

These findings suggest that GEDAE-LaB is a free software easily comprehended by users minimally familiarized with adopted procedures for calculations of energetic profile using oxygen uptake and lactate accumulation during exercise. By providing availability of the software and its source code we hope to facilitate future related research.     

Sperm cryopreservation of Prochilodus brevis using different concentrations of non-permeable cryoprotectants

The action of substances with non-permeable cryoprotectant potential, besides glucose, has not yet been studied for the species Prochilodus brevis. The objective of this work was to evaluate the action of four non-permeable cryoprotectants on this species sperm cryopreservation. Five pools were cryopreserved in a solution of 5% glucose and 10% dimethyl sulfoxide (Me₂SO) associated or not (control) with cryoprotectants egg yolk (5, 10 or 12%), soy lecithin (2.5, 7.5 or 10%), sucrose (5, 10 or 20%) and lactose (5, 8 or 15%). After thawing, samples were evaluated for sperm kinetics (total motility, motility duration, velocities, and wobble - WOB), morphology and membrane and DNA integrity. The treatments containing egg yolk improved significantly (P<0.05) results when compared the control for the membrane integrity parameter. When compared to other treatments, egg yolk, at any concentration, presented higher results (P<0.05) for membrane integrity, total motility, curvilinear velocity (VCL) and average path velocity (VAP) parameters. Egg yolk also showed the best results for WOB, but it did not differ from 5% and 8% lactose and 5% and 20% sucrose. Soy lecithin had the lowest percentages of morphologically normal sperm (P<0.05), while the other treatments did not differ from each other. There was no difference regarding DNA integrity data. Thus, 5% egg yolk is indicated as a non-permeable cryoprotectant for P. brevis, in association with 5% glucose and 10% Me₂SO.     

Chiral discrimination by ligand-exchange chromatography: A comparison between phenylalaninamide-based stationary and mobile phases

The copper(II) complexes of two new diastereomeric ligands, N²-(R)- and N²-(S)-2′-hydroxypropyl-(S)-phenylalaninamide [(R, S)-1 and (S, S)-1], have been used as additives to the eluent in high-performance liquid chromatography (HPLC) reversed phase for the chiral separation of DNS-amino acids. The aim was that of comparing the separation process obtained by the chiral eluent with that obtained by an analogous bonded stationary phase containing (S)-phenylalaninamide, previously studied [CSP-(S)-Phe-NH₂]. The affinity of the ternary complexes for the C₁₈ column was determined by adsorption experiments in HPLC. It was shown that the two systems (chiral eluent, chiral stationary phase) work according to different mechanisms. Ternary complex formation in solution was studied by fluorescence spectroscopy. It was shown that chiral separation with the Cu(II) complexes added to the eluent was determined by the relative affinities of the ternary complexes for the column-stationary phase rather than by their stabilities in solution. With CSP-(S)-Phe-NH₂ the separation is accounted for by the relative stabilities of the ternary complexes, which depends mainly on the “allowed” geometry of the complex and on the steric repulsion of the amino acid side chain with the spacer. © 1996 Wiley-Liss, Inc.     

Kinetic heterogeneity of an experimental tumour revealed by BrdUrd incorporation and mathematical modelling

In the present paper we propose a method of analysis of the cell kinetic characteristics of in vivo experimental tumours, that uses DNA-BrdUrd flow cytometry data at various times after the bromodeoxyuridine (BrdUrd) injection and mathematical modelling. The model of the cell population takes into account the cell-cell heterogeneity of the progression rate across cell cycle phases within the tumour, and assumes a strict correlation between the durations of S and G2M phases. The model also allows for a nonconstant DNA synthesis rate across S phase. In addition, the measurement process is modelled, considering the possibility of nonimpulsive labelling and providing a representation of the time course of the bivariate DNA-BrdUrd fluorescence distribution. Sequential DNA-BrdUrd distributions were obtained in vivo from a human ovarian carcinoma transplanted in mice and, for comparison, in vitro from a cell line of the same origin. From these data, that included the fractional density and the mean BrdUrd-fluorescence of BrdUrd-positive cells as a function of the DNA-fluorescence, kinetic parameters such as the potential doubling time (T _pot) and the mean and variance of the transit times in S and G2M phases, were estimated. This study revealed the presence of a substantial heterogeneity in S and G2M phases within the in vivo cell population and of a lower heterogeneity in the in vitro population. Moreover, our analysis suggests a nonnegligible effect of the BrdUrd pharmacokinetics in the in vivo cell labelling.     

Renal excretion and saline intake during post-stress immobilization period in rats

An experiment in which the rats access either to 0.5% or 1.5% saline was designed in order to further characterise the relationship between sodium intake and renal excretion after acute immobilization stress. A saline solution for 3 days was provided to the rats previous to the experimental day. On that day, after finishing acute immobilization stress, all variables under observation were measured every 6 h for 24 h. These periods were denominated as follows: T1 (12.00 to 18.00 h), T2 (18.00 to 24.00 h), T3 (24.00 to 06.00 h) and T4 (06.00 to 12.00 h). Acute immobilization stress reduced sodium renal excretion in both T1 and T2. Sodium intake in acute immobilization stress rats was lower than in control rats during all observed periods, while the urine volume was only reduced in the stressed animals in T1. These results were similar in both saline solution concentrations. A good correlation was observed between sodium intake and sodium excretion in control rats having access to either 0.5% or 1.5% saline as well as in stressed rats having access to 0.5% saline, this correlation was not observed in stressed rats with 1.5% saline. This suggests that stress impaired the renal capability of rats to handle high sodium but not a slight sodium overload. The inability of the kidney to excrete sodium may be critical to reduce sodium intake after acute immobilization stress.     

Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus

Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca²⁺-mediated signalling. Ca²⁺ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca²⁺ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuB ^KU80, were engineered to express the bioluminescent Ca²⁺-reporter aequorin. An aequorin-mediated method for routine Ca²⁺ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca²⁺]_c) in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca²⁺, oxidative stress or exposure to human serum) that the fungus might be exposed to during infection, were analysed in living conidial germlings. The ‘signatures’ of the transient [Ca²⁺]_c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca²⁺-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca²⁺-signalling components in each stress response. Concordant with the involvement of Ca²⁺-calmodulin complexes in these Ca²⁺-mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca²⁺-signatures to all the challenges. The Ca²⁺-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca²⁺ in initiating the stress responses.