Evaluation of mutagenic, teratogenic, and immunomodulatory effects of Annona nutans hydromethanolic fraction on pregnant mice.

Plants such as Annona nutans used in folk medicine have a large number of biologically active compounds with pharmacological and/or toxic potential. Moreover, pregnant women use these plants indiscriminately, mainly in the form of teas, without being aware of the harm that they could cause to the health of the embryo/fetus. Therefore, it is necessary to analyze the potential toxic effects of medicinal plants during gestation. The present study aimed to evaluate the effects of A. nutans hydromethanolic fraction leaves (ANHMF) on mutagenic and immunomodulatory activity, reproductive performance, and embryo-fetal development in pregnant female mice. The animals (N=50 female and 25 male) were divided into 5 groups: Control, Pre-treatment, Organogenesis, Gestational, and Pre+Gestational. The results indicate that ANHMF mainly contains flavonoid and other phenolic derivatives. It was found that it does not exhibit any mutagenic or immunomodulatory activity, and it does not cause embryo-fetal toxicity. Based on the protocols used in the present studies, our analyses confirm that it is safe to use ANHMF during pregnancy.

A prospective cohort study on post COVID syndrome from a tertiary care centre in Sri Lanka.

There is a scarcity of follow-up data on post-COVID syndrome and its physical, psychological, and quality of life attributes, particularly from South Asian populations. This study was conducted to assess the prevalence, associations, and impact of the post-COVID syndrome among patients treated at a dedicated COVID-19 treatment unit. A prospective cohort study was conducted to follow-up patients with moderate to severe disease or mild disease with co-morbidities at 2 and 6 weeks, 3 and 6 months and 1 year from discharge. Clinical notes, an interviewer-administered questionnaire and six-item cognitive impairment, Montreal Cognitive Assessment, Fatigue (11-item Chalder) and EQ5D5L questionnaires were used for data collection. All patients had follow-up echocardiograms and symptomatic patients had biochemical and haematological investigations, chest x-rays, high-resolution computed tomography of chest and lung function tests. Among 153 patients {mean age 57.2 ± 16.3 years (83 (54.2% males)}, 92 (60.1%) got the severe disease. At least a single post-COVID symptom was reported by 119 (77.3%), 92 (60.1%), 54 (35.3%) and 25 (16.3%) at 6 weeks, 3 months, 6 months and 1 year respectively. Post-COVID symptoms were significantly associated with disease severity (p = 0.004). Fatigue was found in 139 (90.3%), 97 (63.4%) and 66 (43.1%) patients at 2, 6 and 12 weeks respectively. Dyspnoea {OR 1.136 (CI 95% 0.525-2.455)}, arthralgia {OR 1.83(CI 95% 0.96-3.503)} and unsteadiness {OR 1.34 (CI 95% 0.607-2.957)}were strongly associated with age above 60 years. Both genders were equally affected. In multivariable logistic regression, fatigue and anxiety/depression were associated with poor quality of life (QoL) (p = 0.014, p ≤ 0.001) in 6 weeks. In cardiac assessments, diastolic dysfunction (DD) was detected in 110 (72%) patients at 2 weeks and this number reduced to 64 (41.8%) at 12 weeks. The decline in diastolic dysfunction in elderly patients was significantly higher compared to young patients (p = 0.012). Most post-COVID symptoms, QoL and cognition improve during the first few months. The severity of the disease and older age are associated with post-COVID symptoms. Transient DD may contribute to cardiac symptoms of post-COVID syndrome, especially in elderly patients.

Dynamics of the orphan myosin MyoF over Trypanosoma cruzi life cycle and along the endocytic pathway.

Trypanosoma cruzi proliferative forms perform endocytosis through a specialized structure named the cytostome-cytopharynx complex (SPC). The SPC is a specialized invagination of the cell membrane that extends through the cell body towards the posterior regions, with its aperture close to the flagellar pocket. Recently, diverse proteins were found along the cytopharynx, including two myosin motors. One of these is the orphan myosin MyoF, that was proved to be essential for endocytosis in epimastigotes. However, the dynamics of MyoF localization along the endocytic pathway and through the T. cruzi life cycle remain unclear. Using CRISPR-Cas9 genome editing, we generated epimastigotes expressing MyoF fused to mNeonGreen from its endogenous locus. Using these cells, we observed that during the epimastigote cell cycle MyoF signal disappeared during G2, reappearing at early cytokinesis. Additionally, we show that MyoF localization during metacyclogenesis is compatible with the progressive disappearance of the SPC, being absent in metacyclic trypomastigotes. Detergent fractionation showed that MyoF was predominantly present in the insoluble fraction and immunolocalized at the SPC microtubules in whole-mount cytoskeleton preparations. Moreover, during tracer uptake through the SPC, MyoF followed the tracer along the endocytic pathway and was found in posterior compartments after 30 min. Taken together, the data suggest that MyoF may play a role not only at the cargo entry site but also along the endocytic pathway.

Dysregulation of placental ABC transporters in a murine model of malaria-induced preterm labor.

Malaria in Pregnancy (MiP) is characterized by placental accumulation of Plasmodium-infected erythrocytes, intrauterine growth restriction (IUGR) and preterm delivery (PTD). Placental ATP-binding cassette (ABC) transporters mediate the efflux of nutrients, cytokines and xenobiotics. The expression and activity of these transporters are highly responsive to infection. We hypothesized that MiP would perturb the expression of placental ABC transporters, promoting PTD. Peripheral blood, spleens, livers and placentas of pregnant mice, infected with Plasmodium berghei ANKA on gestational day (GD) 13.5, were collected and analyzed on GD18.5. The primary consequences of human MiP, including IUGR, PTD (20%) and placental inflammation, were recapitulated in our mouse model. Electron microscopy revealed attenuated presence of labyrinthine microvilli and dilated spongiotrophoblasts -granular endoplasmic reticulum cisternae. Additionally, a decrease in placental Abca1 (ABCA1), Abcb1b (P-glycoprotein), Abcb9 and Abcg2 (BCRP) expression was observed in MiP mice. In conclusion, MiP associated with PTD impairs placental ABC transporters' expression, potentially modulating placental nutrient, environmental toxin and xenobiotic biodistribution within the fetal compartment, and may, at some degree, be involved with pregnancy outcome in MiP.

Evolutionary conservation of actin-binding proteins in Trypanosoma cruzi and unusual subcellular localization of the actin homologue.

The actin cytoskeleton controls pivotal cellular processes such as motility and cytokinesis, as well as cell-cell and cell-substrate interactions. Assembly and spatial organization of actin filaments are dynamic events regulated by a large repertoire of actin-binding proteins. This report presents the first detailed characterization of the Trypanosoma cruzi actin (TcActin). Protein sequence analysis and homology modelling revealed that the overall structure of T. cruzi actin is conserved and that the majority of amino-acid changes are concentrated on the monomer surface. Immunofluorescence assays using specific polyclonal antibody against TcActin revealed numerous rounded and punctated structures spread all over the parasitic body. No pattern differences could be found between epimastigotes and trypomastigotes or amastigotes. Moreover, in detergent extracts, TcActin was localized only in the soluble fraction, indicating its presence in the G-actin form or in short filaments dissociated from the microtubule cytoskeleton. The trypanosomatid genome was prospected to identify actin-binding and actin-related conserved proteins. The main proteins responsible for actin nucleation and treadmilling in higher eukaryotes are conserved in T. cruzi.

Quantum corrections for the phase diagram of systems with competing order.

We use the effective potential method of quantum field theory to obtain the quantum corrections to the zero temperature phase diagram of systems with competing order parameters. We are particularly interested in two different scenarios: regions of the phase diagram where there is a bicritical point, at which both phases vanish continuously, and the case where both phases coexist homogeneously. We consider different types of couplings between the order parameters, including a bilinear one. This kind of coupling breaks time-reversal symmetry and it is only allowed if both order parameters transform according to the same irreducible representation. This occurs in many physical systems of actual interest like competing spin density waves, different types of orbital antiferromagnetism, elastic instabilities of crystal lattices, vortices in a multigap SC and also applies to describe the unusual magnetism of the heavy fermion compound URu2Si2. Our results show that quantum corrections have an important effect on the phase diagram of systems with competing orders.

A case-control study of protection against tuberculosis by BCG revaccination in Recife, Brazil.

SETTING: Metropolitan region of Recife, Brazil. OBJECTIVE: To estimate the additional protection against tuberculosis (TB) provided by a second dose of bacille Calmette-Guérin (BCG) vaccine. DESIGN: Case-control study. Cases were cases of TB newly diagnosed by the TB control programme, independent of clinical form. Three matched neighbourhood controls were selected using a systematic routine, starting from the case's address. The matching was within the age groups 7-9, 10-14 and 15-19 years. RESULTS: Analysis was conducted among 169 cases and 477 controls. For the efficacy of BCG revaccination against TB overall, matched (crude) vaccine effectiveness (VE) was -3 (95% CI -50-29) and matched (adjusted) VE was 8 (95% CI -77-52). CONCLUSIONS: This study suggests that a second dose of BCG does not offer additional protection. Revaccination should not be offered. As large numbers of subjects are already vaccinated and vaccine appears to offer some protection in older subjects, further studies with larger sample sizes could investigate the potential efficacy of revaccination with BCG in the age group > or = 15 years.

Leishmania-induced tyrosine phosphorylation in the host macrophage and its implication to infection.

Tyrosine phosphorylation is an important mechanism of cell regulation and has been recently implicated in defense strategies against a variety of pathogens. We have investigated the involvement of protein tyrosine kinase activity in the Leishmania attachment, invasion and survival within macrophages, as well as promastigote ability to trigger tyrosine phosphorylation, which could contribute to leishmanicidal activity. Treatment of murine macrophage monolayers with genistein, herbimycin A, tyrphostin 25 or staurosporine prior to infection decreased parasite invasion in a dose-dependent manner. Contrary, addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, phosphotyrosine and p-nitrophenyl phosphate to the interaction medium, significantly increased parasite binding and internalization, whereas phosphoserine and phosphothreonine had no effect. The phosphatase activity of intact promastigotes was greater than that of macrophages. Western blot analysis revealed tyrosine-phosphorylated bands from 198 to 28 kDa following macrophage challenge with promastigotes. Uninfected macrophages displayed no detectable tyrosine phosphorylated proteins, possibly indicating an inducible process, while in parasites it was constitutive, as seen by the presence of 42, 40 and 35 kDa phosphoproteins on the Leishmania lysates. Immunofluorescence and immunogold detection of phosphotyrosine residues in some promastigote-macrophage attachment areas, but not in the vicinity of ingested parasites, suggest that Leishmania-induced tyrosine phosphorylation is an early, local and short-lived event. Genistein treatment of Leishmania-infected cells significantly enhanced the parasite burden. This antagonist also diminished nitric oxide production in resting and interferon gamma/lipopolysaccharide-activated infected macrophages, which may account for the increased parasite survival. We propose that protein tyrosine kinase-linked pathways regulate the Leishmania promastigote invasion and the macrophage microbicidal activity.

Isolation and biochemical characterization of the costa of Tritrichomonas foetus.

The costae are cytoskeletal structures found in Trichomonadidae. Both the structural organization and composition of this organelle are still unknown. In the present work we have introduced a new methodology for the costa isolation. Using sucrose density-gradient centrifugation an enriched costa fraction was obtained. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the costa contains several proteins, with major bands corresponding to apparent molecular masses of 122, 115, 112, 93, 87, 82, 59, 52, 44, 41, 32, and 26 kDa. No significant amount of carbohydrates was detected in the costa fraction. The fractionation methodology described here has the advantage of using normal centrifugation methods and is being applied to trichomonas in the size range of Tritrichomonas foetus and Trichomonas vaginalis.

Isolation and characterization of a highly purified flagellar membrane fraction from trypanosomatids.

The flagellar membrane of trypanosomatids has certain ultrastructural and antigenic characteristics that make its biochemical analysis very interesting. We have obtained a highly purified flagellar membrane fraction from epimastigotes of Trypanosoma cruzi and promastigotes of Herpetomonas samuelpessoai. The fractions consisted of regular spherical vesicles. Membrane fractions fixed in a glutaraldehyde solution containing filipin and freeze-fractured showed few intramembranous particles, and many protuberances indicative of filipin-sterol complexes. The protein composition of all the subflagellar fractions was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The flagellar membrane fraction showed more than twenty bands. We wish particularly to point out the presence of six bands corresponding to proteins of 160, 135, 41, 31, 26 and 18 kDa which were not present in the axoneme-paraxial structure fraction. The blot of flagellar membrane proteins successively incubated with concanavalin A and horseradish peroxidase showed intense binding of the lectin to proteins of 117 and 87 kDa, and less strong binding to several other minor bands. Our results showed that although the membrane of the flagellum of trypanosomatids had few proteins, it seemed rich in glycosylated elements.

Purification of the paraflagellar rod of the trypanosomatid Herpetomonas megaseliae and identification of some of its minor components.

The paraflagellar rod (PFR) is a component of the flagellar cytoskeleton of trypanosomatid protozoa, representing a filamentous structure that runs alongside the common 9 + 2 microtubular axoneme. The high degree of ultrastructural complexity and organization of the PFR suggests that it might be formed by numerous biochemical components. However, biochemical analysis of the PFR has revealed, to date, a modest degree of complexity in what concerns both major and minor PFR proteins. In this paper the preparation of purified PFR fractions by a combination of conventional cell-fractionation procedures, non-ionic detergent treatment and limited proteolysis is described. Comparative SDS-PAGE analysis of the different purification steps indicates that the purified PFR fractions possess high amounts of the well-known major PFR proteins (77 and 83 kDa). Also, bands of 147, 139, 129 and 122 kDa are clearly enriched in such fractions and may correspond to minor PFR components. A slight enrichment in a specific fraction of a doublet of bands of 181/188 kDa suggest the participation of these proteins in the composition of the bridges between the PFR and the axoneme.

Synthesis of vitellogenin by the follicle cells of Rhodnius prolixus.

The synthesis and secretion of vitellogenin by the ovary of Rhodnius prolixus was investigated. Using whole ovary or epithelial cells isolated from follicles of different sizes, it is shown that the follicle cells are a site of synthesis for this protein in the ovary. The ovaries or follicle cells were incubated in vitro with [(35)S]-methionine or (32)Pi and the secretion of newly synthesized ovarian vitellogenin (O-Vg) was estimated by the radioactivity associated with the immunoprecipitate or acid-precipitate proteins in the culture medium. The radioactive O-Vg was analyzed by SDS-PAGE followed by autoradiography or after elution from a DEAE-Toyopearl column. The presence of O-Vg inside the follicle cells was detected by immunofluorescence and immunogold labels. Both methods revealed strong labeling inside the follicle cells. While the capacity for total protein synthesis by the follicle cells was maximal during the early phase of vitellogenesis (in small follicles), the synthesis of O-Vg reached its peak during the late phase of oocyte growth, just before formation of the chorion. A possible role for ovarian vitellogenin in Rhodnius and its relationship with Vg synthesis by the fat body is discussed.

Intracellular studies of dopamine neurons in vitro: pacemakers modulated by dopamine.

Intracellular recordings from dopamine (DA)-sensitive neurons in rat substantia nigra tissue slices revealed that these neurons exhibit spontaneous pacemaker-like activity. DA-sensitive neurons had higher input resistances, larger time constants and less linear voltage responses to current injection than did non-DA-sensitive neurons in the zona compacta. The administration of DA produced an inhibition of firing rate, a hyperpolarization and a decrease in input resistance. These effects were blocked by (-)sulpiride, a selective D2 antagonist. A reversal potential of -88 +/- 14 mV was calculated for the DA-induced hyperpolarization suggesting the involvement of potassium ions in the mechanism of DA action.

Neuronal sensitivities in preoptic tissue slices: interactions among homeostatic systems.

The preoptic area participates in many homeostatic systems, which include the regulation of body temperature, fluid and metabolite balance, and reproduction. Some preoptic neurons have been shown to be sensitive to either temperature, osmotic pressure, glucose, testosterone or estradiol. While previous studies have treated these as separate and distinct neuronal populations, this paper reviews recent experiments which show that many neurons have multiple sensitivities to these endogenous factors. Neurons in preoptic tissue slices were tested for their responses to changes in temperature, as well as various perfusion media containing 30 pg/ml testosterone or estradiol, low glucose (1.0 mM) or increased osmotic pressure (309 mosmol/kg). The steroid-sensitive, osmosensitive and glucosensitive neurons were not confined to the temperature insensitive neurons; but instead nearly half of the thermosensitive neurons responded to these nonthermal stimuli. In addition, many osmosensitive neurons showed glucosensitivity and steroid-sensitivity. This suggests that, even at the neuronal level, there is a basis for interactions between homeostatic systems.

A slice chamber for intracellular and extracellular recording during continuous perfusion.

The design of a tissue slice perfusion system is described, and examples are given showing the stability of this system for intracellular and extracellular recordings during changes in perfusion media. The stability of this system is attributed to several features. Mini-drips serve to cushion transient changes in flow rate when switching from one medium to another. Solenoid valves are used to quickly switch perfusion media with minimal mechanical movement. A finely-controlled adjustable flow valve provides a uniform flow rate for all media. Constant tissue temperature is maintained by media perfusion through a thermoelectric Peltier assembly. In addition, a filter paper wick insures that the perfusate is constantly removed without movement in the tissue slices. With this design, the slices are supported on a net at the interface between the perfusion medium and a humidified, oxygenated atmosphere. This arrangement appears to be conducive to tissue viability and facilitates the placement of microelectrodes in the slices.

Oxygen consumption of hypothalamic tissue slices after varying incubation periods.

In order to quantitate possible time-related changes in the viability of rat hypothalamic tissue slices, tissue oxygen consumption was measured after incubation periods ranging from 0-4 hours. There were no significant differences in mean tissue oxygen consumption between the various incubation periods; nor was there any trend indicating that oxygen consumption gradually decreases over time. Moreover, no regional differences were observed among the various rostral hypothalamic slices. One obvious trend, however, was that during the first two hours of each experiment, tissue oxygen consumption decreased briefly and then returned to normal higher levels. The exact occurrence of this transitory decrease varied from experiment to experiment; but the subsequent recovery in oxygen consumption was always complete by two hours of incubation. This initial transient decrease in tissue oxygen consumption may reflect the initial period of electrophysiological inactivity reported in several in vitro studies.

Individual housing from rearing modifies the performance of young rats on the elevated plus-maze apparatus.

Previous studies from our laboratory have demonstrated that male and female rats exhibit a differential pattern of behavior in the elevated plus-maze as a function of age. In the present study, the influence of individual housing conditions on young animals treated with one of two classical anxioselective drugs, diazepam or pentylenetetrazole, was investigated in the elevated plus-maze. In Experiment I, males and females were housed for 30 days after weaning either individually or in groups, and tested in the elevated plus-maze at 60 days of age. In Experiment 2, the effects of diazepam (0.75 or 1.0 mg/kg) or of pentylenetetrazole (20 or 30 mg/kg) on the behavior of isolated or grouped rats were studied at 60 days of age in the elevated plus-maze. The results show that isolated housed animals tested with diazepam at 60 days of age exhibited increased frequency and time spent on the open arms of the apparatus compared to control rats. The effect of diazepam was not observed in grouped animals tested at 60 days of age. Pentylenetetrazole produced a decrease in the frequency and time spent on the open arms. This effect was more prominent in grouped animals. The results suggest that 60-day-old rats deprived of playfighting experience present high basal anxiety levels and also that rearing conditions (isolated or grouped) are able to interact with both anxiolytic and anxiogenic effects of experimental drugs.

Effects of potassium on the ethanol production rate of Saccharomyces cerevisiae carrying the plasmid pCYG4 related with ammonia assimilation.

The influence of potassium on ethanol production by Saccharomyces cerevisiae wild type and AR5 cells carrying the plasmid pCYG4 was investigated. This plasmid carries the glutamate dehydrogenase gene conferring an 11-fold higher level of expressed enzyme activity over the wild type cells. All experiments were carried out in batch culture with medium supplemented to different potassium concentrations up to 180 mM. Maximum ethanol production rate was observed in the AR5 cells grown in medium supplemented with 3.5 mM of potassium ions. Glucose uptake rate increased with increasing potassium up to 60 mM, but higher concentrations depressed glucose uptake rate in both strains. Furthermore, the wild type cells showed higher growth rate, ethanol production, and glucose consumption rate than the AR5 cells. These lower rates in the AR5 cells could be explained by repression of potassium uptake by an enhancement of ammonium feeding, and greater energy requirements by these cells due the presence of the plasmid.

Silagem de híbridos de sorgo associado a percentagens de concentrado no desempenho de novilhos / Effect of sorghum hybrids silage associated to percentage of concentrate levels on the performance of steers

Avaliou-se o desempenho de novilhos alimentados em confinamento com silagem de híbridos de sorgo forrageiro e de duplo propósito associado a níveis de concentrado. Foram utilizados 60 novilhos, com idade inicial de 20 meses e peso corporal inicial de 322,45±5,58kg, distribuídos inteiramente ao acaso, nos seguintes tratamentos: três porcentagens de concentrado e dois tipos de silagem de sorgo. Foi verificada interação tipo de silagem versus porcentagem concentrado para consumo de matéria seca e ganho de peso médio diário, os quais aumentaram com o aumento de concentrado na dieta com silagem de sorgo forrageiro, mas não com silagem de sorgo duplo propósito. Os novilhos alimentados com silagem de sorgo forrageiro consumiram menos energia digestível (27,89Mcal/dia-1) que aqueles alimentados com silagem de sorgo duplo propósito (31,42Mcal/dia-1). A conversão alimentar média não foi alterada pelo tipo de silagem utilizada. O aumento da porcentagem de concentrado em dietas com volumoso de menor qualidade aumenta a eficiência biológica de bovinos confinados.

The feedlot performance of steers fed forage sorghum and double purpose hybrids silage associated with concentrate levels was evaluated. A total of 60 steers, initial age 20 months and body weight of 322.45±5.58kg, were randomized in the treatments: three concentrate levels and two types of sorghum silage. The interaction of sorghum silage versus concentrate levels was observed for dry matter intake and average daily weight gain, which increased with the increase concentrate level in diets with forage sorghum silage, but was not altered in diets with double purpose silage. Steers fed forage sorghum silage presented a lower average intake of digestible energy (27.89Mcal/day-1) than those fed double purpose sorghum silage (31.42Mcal/day-1). The average feed conversion was not affected by the type of silage used. The increase of concentrate level in diets with lower forage quality increases the biological efficiency of feedlot beef cattle.

Interactions of reproductive steroids, osmotic pressure, and glucose on thermosensitive neurons in preoptic tissue slices.

The preoptic area contains thermosensitive neurons, thought to be important in thermoregulation, and steroid-sensitive neurons, thought to be involved in reproduction. The preoptic area also contains osmosensitive neurons, considered important in water balance, and glucosensitive neurons, thought to function in the regulation of glucose. If these various neurons belong to separate populations, one might predict that most osmosensitive, glucosensitive, and steroid-sensitive neurons constitute the population of temperature-insensitive neurons rather than thermosensitive neurons. To test this hypothesis, single unit activity was recorded in preoptic tissue slices prepared from male rats. In addition to temperature changes, neuronal responses were examined with various perfusion media containing testosterone or estradiol (30 pg/mL), low glucose (1.0 mM), and increased osmotic pressure (309 mosmol/kg). It was found that the steroid-sensitive, osmosensitive, and glucosensitive neurons were not confined to the temperature-insensitive neurons; but that nearly half of the thermosensitive neurons responded to these nonthermal stimuli. This lack of specificity was also observed between osmosensitive and glucosensitive neurons; however, most of the steroid-sensitive neurons were highly specific for either estradiol or testosterone. Although these findings do not suggest a strong functional specificity for preoptic neurons, they do support studies emphasizing interactions between regulatory systems.