Evolution of CpG Islands within the myc Gene Family

CpG islands are discrete regions of DNA with significantly greater frequencies of CpG doublets than bulk genomic DNA. They are most frequently associated with the 5′-ends of housekeeping genes and are involved in the regulation of their expression. In this study, the structure and evolution of CpG islands within genes of the myc family were evaluated with the protein-coding sequences of animals and their transducing viruses. These evaluations relied on a gene tree for the entire myc family to test the origins of CpG islands within their two protein-coding exons. Overall, CG-very rich and CG-rich islands are associated with exon 2 of the different myc genes of warm-blooded vertebrates and with exon 3 of the N-myc and s-myc sequences of mammals, but not birds. These overall distributions of well-developed islands can be related to the major transitions of the CG-rich genomes of warm-blooded vertebrates from the CG-poor ones of other animals. In turn, the greater variability of well-developed islands within exon 3 of the N-myc gene and among the different retrogenes of the myc family can be attributed to their reduced functional constraints, as evidenced by their limited and very restricted patterns of expression, respectively.     

Brazilian sleeping beauty: First evidence of hibernation by a bat in South America

Torpor and hibernation are energy-saving heterothermic strategies widespread among mammals, and bats have reports of hibernation in North America, Europe, Asia, Oceania, and Africa. However, to date, there was no evidence of hibernation by bats in South America. We tagged four individuals of Eptesicus brasiliensis and one remained torpid for 13 consecutive days in a tree cavity roost in southern Brazil, showing evidence that bats can hibernate in South America.     

Multifrequency calorimetry of the folding/unfolding transition of cytochrome c.

The folding-unfolding transition of Fe(III) cytochrome c has been studied with the new technique of multifrequency calorimetry. Multifrequency calorimetry is aimed at measuring directly the dynamics of the energetic events that take place during a thermally induced transition by measuring the frequency dispersion of the heat capacity. This is done by modulating the folding/unfolding equilibrium using a variable frequency, small oscillatory temperature perturbation (approximately 0.05-0.1 degrees C) centered at the equilibrium temperature of the system. Fe(III) cytochrome c at pH 4 undergoes a fully reversible folding/unfolding transition centered at 67.7 degrees C and characterized by an enthalpy change of 81 kcal/mol and heat capacity difference between unfolded and folded states of 0.9 kcal/K*mol. By measuring the temperature dependence of the frequency dispersion of the heat capacity in the frequency range of 0.1-1 Hz it has been possible to examine the time regime of the enthalpic events associated with the transition. The multifrequency calorimetry results indicate that approximately 85% of the excess heat capacity associated with the folding/unfolding transition relaxes with a single relaxation time of 326 +/- 68 ms at the midpoint of the transition region. This is the first time that the time regime in which heat is absorbed and released during protein folding/unfolding has been measured.     

The fully-active and structurally-stable form of the mitochondrial ATP synthase of Polytomella sp. is dimeric

Mitochondrial F₁F_O-ATP synthase of chlorophycean algae is a stable dimeric complex of 1,600 kDa. It lacks the classic subunits that constitute the peripheral stator-stalk and the orthodox polypeptides involved in the dimerization of the complex. Instead, it contains nine polypeptides of unknown evolutionary origin named ASA1 to ASA9. The isolated enzyme exhibited a very low ATPase activity (0.03 Units/mg), that increased upon heat treatment, due to the release of the F₁ sector. Oligomycin was found to stabilize the dimeric structure of the enzyme, providing partial resistance to heat dissociation. Incubation in the presence of low concentrations of several non-ionic detergents increased the oligomycin-sensitive ATPase activity up to 7.0–9.0 Units/mg. Incubation with 3% (w/v) taurodeoxycholate monomerized the enzyme. The monomeric form of the enzyme exhibited diminished activity in the presence of detergents and diminished oligomycin sensitivity. Cross-linking experiments carried out with the dimeric and monomeric forms of the ATP synthase suggested the participation of the ASA6 subunit in the dimerization of the enzyme. The dimeric enzyme was more resistant to heat treatment, high hydrostatic pressures, and protease digestion than the monomeric enzyme, which was readily disrupted by these treatments. We conclude that the fully-active algal mitochondrial ATP synthase is a stable catalytically active dimer; the monomeric form is less active and less stable. Monomer-monomer interactions could be mediated by the membrane-bound subunits ASA6 and ASA9, and may be further stabilized by other polypeptides such as ASA1 and ASA5. Alexa Villavicencio-Queijeiro and Miriam Vázquez-Acevedo have contributed equally to this work.     

Bottom-up advocacy strategies to abortion access during the COVID-19 pandemic: Lessons learned towards reproductive justice in Brazil

In Brazil, abortion is only allowed in cases of rape, serious risk to a woman's life or fetal anecephaly. Legal abortion services cover less than 4% of the Brazilian territory and only 1,800 procedures are performed, in average, per year. During the COVID-19 pandemic, almost half of the already few Brazilian abortion clinics shut down and women had to travel even longer distances, reaching abortion services at later gestational ages. In this paper, we describe three bottom-up advocacy strategies that emerged from difficulties deepened during the COVID-19 pandemic at a single abortion service in Brazil, amidst anti-gender policies from the federal government. Telemedicine abortion, outpatient surgical abortion and the provision of abortion after 20 weeks' gestation are important strategies that may reduce inequalities that impact the most vulnerable populations, such as black and indigenous women, children, adolescents and women experiencing domestic violence.     

Calorimetric determination of the energetics of the molten globule intermediate in protein folding: apo-alpha-lactalbumin

High-sensitivity differential scanning calorimetry has been used to characterize the energetics of the molten globule state of apo-alpha-lactalbumin. This characterization has been possible by performing temperature scans at different guanidine hydrochloride (GuHCl) concentrations in order to experimentally define the temperature-GuHCl stability surface of the protein. Multidimensional analysis of the heat capacity surface has allowed simultaneous resolution of the energetics of the unfolded and molten globule states. These experiments indicate that the intrinsic enthalpy difference (i.e., excluding additional contributions such as those arising from differential GuHCl binding) between the unfolded and native states is 31.8 kcal/mol at 25 degrees C whereas that of the molten globule and native states is only 7.7 kcal/mol. At the same temperature, the entropy changes are 99.2 and 23.7 cal/K.mol and the heat capacity changes are 1821 and 326 cal/K.mol, respectively. Analysis of the thermodynamic data indicates that in passing from the native to the molten globule state only approximately 19% of the hydrogen bonds are broken. In addition, the magnitude of delta Cp for the molten globule suggests that water does not largely penetrate into the interior of the molten globule, implying that significant hydrophobic interactions are still present in this state. These parameters provide precise energetic constraints to the allowed structural conformations of the molten globule.     

Fluorescence energy transfer in two dimensions. A numeric solution for random and nonrandom distributions.

A method of Monte Carlo calculations has been applied to the problem of fluorescence energy transfer in two dimensions in order to provide a quantitative measure of the effects of nonideal mixing of lipid and protein molecules on the quenching profiles of membrane systems. These numerical techniques permit the formulation of a detailed set of equations that describes in a precise manner the quenching and depolarization properties of planar donor-acceptor distributions as a function of specific spectroscopic and organizational parameters. Because of the exact nature of the present numeric method, these results are used to evaluate critically the validity of previous approximate treatments existing in the literature. This method is also used to examine the effects of excluded volume interactions and distinct lattice structures on the expected transfer efficiencies. As a specific application, representative quenching profiles for protein-lipid mixtures, in which donor groups are covalently linked to the protein molecules and acceptor species are randomly distributed within lipid domains, have been obtained. It is found that the existence of phase-separated protein domains gives rise to a shielding effect that significantly decreases the transfer efficiencies with respect to those expected for an ideal distribution of protein molecules. The results from the present numerical study indicate that the experimental application of fluorescence energy transfer measurements in multicomponent membrane systems can be used to obtain organizational parameters that accurately reflect the lateral distribution of protein and lipid molecules within the bilayer membrane.     

Mating and role of seminal receptacles in the reproductive biology of the spider crab Maja squinado (Decapoda, Majidae)

The reproductive biology of the spider crab Maja squinado was analyzed based on monthly samples from an 18-month study carried out in Galicia (NW Spain) and laboratory experiments holding primiparous and multiparous females in captivity with and without males. The seminal receptacles of adult females were analyzed and their relationship with the presence and developmental stage of the eggs and the gonad maturity stage was determined. Gonad maturation in primiparous females began one or two months after the pubertal moult. Females having gonads in an advanced stage of development made their appearance in December and the first spawning took place in mid-winter or early spring. The percentage of ovigerous females from March to September was ∼75%. As the incubation period progressed, the ovaries became mature again in order to carry out the next spawning. Under experimental conditions the breeding cycle started earlier in multiparous females, during their second yearly cycle, than in primiparous ones. After mating, female spider crabs store sperm in seminal receptacles and this sperm is used in the fertilization of eggs immediately prior to spawning. The analyses of seminal receptacles consisted of the estimation of fullness and the number of differentiated sperm masses. The number of masses ranged between 0 and 6 in field samples (median for females with stored sperm=1) and was positively correlated with fullness. Differences in colour and volume of individual masses showed that, at least in some cases, females carried out successive matings with long intervals in between. This storage mechanism allowed females to fertilize successive broods without remating (as was also shown under experimental conditions). Juvenile females from shallow waters did not have developed seminal receptacles which indicated that mating was not possible until the onset of maturity. Postpubertal females in shallow waters (August to October), including animals participating in aggregations, always showed empty receptacles. The seasonality of receptacle fullness showed that mating involved hard-shelled females and occurred in deep water during the autumn migration from juvenile habitats or in the wintering habitats, during the last stages of gonad maturation (November to February). After fertilization ovigerous females continued to store sperm, but the volume was lower than in non-ovigerous females. Mating may occur in ovigerous females, particularly in the final period of incubation, because in females with broods almost ready to hatch, both new and older sperm masses were seen in the receptacles (distinguished by colour and size). The fullness of the receptacles decreased both in ovigerous and non-ovigerous females in the final phase of the annual breeding cycle (August–October), however, some sperm was still available. In the laboratory, mating was observed, and no courtship nor postcopulatory guarding was recorded. The analysis of receptacles from laboratory experiments indicated that primiparous and multiparous females showed differences in the seasonality of mating in the first phase of the breeding cycle (September–January), related to differences in the timing of gonad maturation and hatching. Mating occurred in the final stages of gonad maturation, a short time before hatching, and matings were detected in ovigerous females. Multiple matings were also evident, to a greater extent than in the field, probably due to the higher availability of males. Females underwent over four successive spawnings in the laboratory without having to recopulate, and the incubation lasted on the average from 40 to 58 days (∼18 and 16°C respectively) and the mean duration between hatching and the next spawning was 3.4 days. It is estimated that most females carry out three successive spawnings during the annual cycle.     

In situ kinetic analysis of glyoxalase I and glyoxalase II in Saccharomyces cerevisiae.

The kinetics of glyoxalase I [(R)-S-lactoylglutathione methylglyoxal-lyase; EC 4.4.1.5] and glyoxalase II (S-2-hydroxyacylglutathione hydrolase; EC 3.1.2.6) from Saccharomyces cerevisiae was studied in situ, in digitonin permeabilized cells, using two different approaches: initial rate analysis and progress curves analysis. Initial rate analysis was performed by hyperbolic regression of initial rates using the program HYPERFIT. Glyoxalase I exhibited saturation kinetics on 0.05-2.5 mM hemithioacetal concentration range, with kinetic parameters Km 0.53 +/- 0.07 mM and V (3.18 +/- 0.16) x 10(-2) mM.min(-1). Glyoxalase II also showed saturation kinetics in the SD-lactoylglutathione concentration range of 0.15-3 mM and Km 0.32 +/- 0.13 mM and V (1.03 +/- 0.10) x 10(-3) mM.min(-1) were obtained. The kinetic parameters of both enzymes were also estimated by nonlinear regression of progress curves using the raw absorbance data and integrated differential rate equations with the program GEPASI. Several optimization methods were used to minimize the sum of squares of residuals. The best parameter fit for the glyoxalase I reaction was obtained with a single curve analysis, using the irreversible Michaelis-Menten model. The kinetic parameters obtained, Km 0.62 +/- 0.18 mM and V (2.86 +/- 0.01) x 10(-2) mM.min(-1), were in agreement with those obtained by initial rate analysis. The results obtained for glyoxalase II, using either the irreversible Michaelis-Menten model or a phenomenological reversible hyperbolic model, showed a high correlation of residuals with time and/or high values of standard deviation associated with Km. The possible causes for the discrepancy between data obtained from initial rate analysis and progress curve analysis, for glyoxalase II, are discussed.