Alpha lipoic acid controls degeneration and ensures follicular development in ovine ovarian tissue cultured in vitro.

This study aims to evaluate the effects of adding alpha lipoic acid (ALA) to the in vitro ovarian tissue culture medium, either fresh or after vitrification/warming. For this purpose, 10 ovaries from five adult sheep were used. Each pair of ovaries gave rise to 16 fragments and were randomly distributed into two groups: fresh (n = 8) and vitrified (n = 8). Two fresh fragments were fixed immediately and considered the control, while another six were cultured in vitro for 14 days in the absence; presence of a constant (100 µM/0-14 day) or dynamic (50 µM/day 0-7 and 100 µM/day 8-14) concentration of ALA. As for the vitrified fragments, two were fixed and the other six were cultured in vitro under the same conditions described for the fresh group. All the fragments were subjected to morphological evaluation, follicular development and stromal density (classical histology), DNA fragmentation (TUNEL), senescence (Sudan Black), fibrosis (Masson's Trichome), and endoplasmic reticulum stress (immunofluorescence). Measurements of the antioxidant capacity against the free radicals 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and estradiol (E2) levels in the culture medium was performed. The results showed that in the absence of ALA, in vitro culture of vitrified ovarian fragments showed a significant reduction (P < 0.05) in follicular morphology and increased the presence of senescence and tissue fibrosis (P < 0.05). Dynamic ALA maintained E2 levels unchanged (P > 0.05) until the end of vitrified ovarian tissue culture and controlled the levels of ABTS and DPPH radicals in fresh or vitrified cultures. Therefore, it is concluded that ALA should be added to the vitrified ovarian tissue in vitro culture medium to reduce the damage that leads to loss of ovarian function. To ensure steroidogenesis during in vitro culture, ALA should be added dynamically (different concentrations throughout culture).

Maternal supplementation with docosahexaenoic acid does not cause constriction of fetal ductus arteriosus: randomized controlled trial.

OBJECTIVE: Docosahexaenoic acid (DHA) is recommended routinely in pregnancy to promote fetal development. DHA has anti-inflammatory activity, but its effects on the fetal heart and circulation are unknown. This study aimed to investigate whether maternal DHA supplementation in the third trimester affects maternal prostaglandin levels and fetal ductus arteriosus flow dynamics. METHODS: This was a double-blind randomized controlled trial with parallel groups conducted between 2018 and 2021. Pregnant women aged over 18 years with a normal fetus at 27-28 weeks' gestation showing no cardiac/extracardiac anomalies or ductal constriction were eligible for the trial. Women consuming substances with a known inhibitory effect on prostaglandin metabolism, such as non-steroidal anti-inflammatory drugs and polyphenol-rich foods, were excluded. The intervention group received oral supplementation of omega-3 with 450 mg/day of DHA for 8 weeks and the placebo group received capsules of soy lecithin for 8 weeks. Anthropometric measurements, assessment of polyphenol and omega-3 consumption, fetal morphological ultrasound examination, fetal Doppler echocardiographic examination and blood sample collection were performed at the start of the study and the latter two were repeated at follow-up. Prostaglandin E2 (PGE2) level and echocardiographic parameters were compared between the intervention and placebo groups and between baseline and follow-up. RESULTS: A total of 24 participants were included in each group. After 8 weeks, there were no significant differences between the intervention and placebo groups in maternal serum PGE2 level or Doppler echocardiographic parameters of ductal flow. No case of ductus arteriosus constriction was observed. The expected intragroup changes in cardiac morphology, as a result of advancing gestation, were present. CONCLUSIONS: Maternal DHA supplementation in the third trimester at a clinically recommended dose did not result in inhibition of PGE2 or constriction of the ductus arteriosus. These findings should be confirmed in postmarket surveillance studies with larger patient numbers in order to test the full safety profile of DHA and provide robust clinical reassurance. © 2024 International Society of Ultrasound in Obstetrics and Gynecology.

Parkia from Cerrado: phytochemical bioprospection, toxicity and in vitro bioactivities of bark and flower extracts.

Parkia platycephala is the only species of the genus Parkia that is endemic to the brazilian Cerrado and the tree symbol of the state of Tocantins, but there are still few studies regarding its bioprospecting. In this study, we aimed to investigate the phytochemical composition, toxicity and bioactivities of the bark and flower of Parkia platycephala. Hot sequential extractions (Soxhlet) were performed using methanol and hydroethanolic solution (70%), after degreasing the sample (hexane). The presence of flavonoids, tannins, steroids and alkaloids was detected in the preliminary screening. Trilinolein, (Z)-9-octadecenamide, 3-O-methyl-d-glucose were detected by Gas Chromatography coupled to Mass Spectrometry (GC-MS). In the Liquid Chromatography with Diode Array Detector (LC-PDA) analysis, it was detected exclusively ferulic acid (bark) and ellagic acid (flower). The ethanolic extract of the bark (IC50=10.69 ± 0.35 µgmL-1) has an antioxidant potential (DPPH• radical) higher than that of the rutin standard (IC50=15.85 ± 0.08 µgmL-1). All extracts showed excellent anticholinesterase potential (Ellman), with emphasis on the ethanol extract of the flower (IC50 =5.34 ± 0.12 µgmL-1). Regarding toxicity (Artemia salina), the methanolic extract of the bark and the ethanolic extract of the flower presented high and moderate levels, respectively. Such results limit the concentrations of biological activities in this study, however, the antioxidant and anticholinesterase indices fall short of toxicity. The results demonstrated promising antioxidant and anticholinesterase activities of both the bark and the flower of Parkia platycephala.

Effect of carvacrol antioxidant capacity on oocyte maturation and embryo production in cattle.

Carvacrol (C10H14O), an efficient phenolic antioxidant substance for several cell types, may become a useful antioxidant for female germ cells and embryo culture. This study investigates the effects of carvacrol supplementation on bovine oocytes in in vitro maturation (IVM) and embryo production. In total, 1222 cumulus-oocyte complexes were cultured in TCM-199+ alone (control treatment) or supplemented with carvacrol at the concentrations of 3 µM (Carv-3), 12.5 µM (Carv-12.5), or 25 µM (Carv-25). After IVM, the oocytes were subjected to in vitro fertilization and embryo production, and the spent medium post-IVM was used for evaluating the levels of reactive oxygen species and the antioxidant capacity (2,2-diphenyl-1-picryl-hydrazyl-hydrate and 2,2'-azinobis-3-ethyl-benzothiozoline-6-sulphonic acid quantification). A greater (P < 0.05) antioxidant potential was observed in the spent medium of all carvacrol-treated groups compared with the control medium. Moreover, the addition of carvacrol to the maturation medium did not affect (P > 0.05) blastocyst production on days 7 and 10 of culture; however, the total number of cells per blastocyst was reduced (P < 0.05) in two carvacrol-treated groups (Carv-3 and Carv-25). In conclusion, carvacrol demonstrated a high antioxidant capacity in the spent medium after oocyte maturation; however, although embryo production was not affected, in general, carvacrol addition to IVM medium reduced the total number of cells per blastocyst. Therefore, due to the high antioxidant capacity of carvacrol, new experiments are warranted to investigate the beneficial effects of lower concentrations of carvacrol on embryo production in cattle and other species.

Oocyte in vitro maturation with eugenol improves the medium antioxidant capacity and total cell number per blastocyst.

This study investigates the impact of eugenol (EU) supplementation on bovine oocyte in vitro maturation (IVM) and antioxidant capacity, as well as in vitro embryo production and quality after conventional in vitro fertilization (IVF). A total of 1077 cumulus oocyte complexes were cultured in TCM-199+ without EU supplementation (control treatment) or supplemented with EU at the concentrations of 10 µM (EU-10), 20 µM (EU-20), or 40 µM (EU-40). After IVM, the oocytes were subjected to IVF and embryo culture. The addition of EU at 40 µM to the IVM medium improved (P < 0.05) the antioxidant capacity and cleavage rate when compared to the control treatment. Moreover, a positive correlation (r = 0.61, P < 0.03) was observed between cleavage rate and EU concentration. The addition of EU at concentrations of 10 and 20 µM decreased (P < 0.05) the calreticulin (CALR) levels in expanded blastocysts when compared to the control treatment and EU-40 treatment. However, the EU-10 and EU-20 treatments had a greater (P < 0.05) mean total cell number (TCN) per expanded blastocyst when compared to the control treatment and EU-40 treatment. In conclusion, the addition of EU to the enriched culture medium during IVM of bovine oocytes improved the antioxidant capacity of the spent medium, as well as the cleavage rate and embryonic quality (i.e., TCN/expanded blastocyst), and reduced the endoplasmic reticulum stress (i.e., CALR levels) in the embryos. Thus, we recommend enriching the IVM medium with 10 µM EU for in vitro bovine embryo production.

Eugenol Improves Follicular Survival and Development During in vitro Culture of Goat Ovarian Tissue.

This study evaluated the effects of different concentrations (10, 20, or 40 µM) of eugenol (EUG 10, EUG 20, or EUG 40), ascorbic acid (50 µg/mL; AA) or anethole (300 µg/mL; ANE 300) on the in-vitro survival and development of goat preantral follicles and oxidative stress in the cultured ovarian tissue. Ovarian fragments from five goats were cultured for 1 or 7 days in Alpha Minimum Essential Medium (α-MEM+) supplemented or not with AA, ANE 300, EUG 10, EUG 20 or EUG 40. On day 7 of culture, when compared to MEM, the addition of EUG 40 had increased the rate of follicular development, as observed by a decrease in the proportion of primordial follicles alongside with an increase in the rate of normally developing follicles. Furthermore, EUG 40 significantly increased both follicular and oocyte diameters. Subsequently, ovarian fragments from three goats were cultured for 1 or 7 days in α-MEM+ supplemented or not with AA, ANE 300 or EUG 40. All tested antioxidants, except ANE 300, were able to significantly decrease the levels of reactive oxygen species in the ovarian tissue, but EUG 40 could most efficiently neutralize free radicals. All ovarian tissues cultured in the presence of antioxidants, especially EUG 40, presented a significant decrease in H3K4me3 labeling, indicating a silencing of genes that play a role in the inhibition of follicular activation and apoptosis induction. When compared to cultured control tissues, both EUG 40 and ANE 300 significantly increased the intensity of calreticulin labeling in growing follicles. The mRNA relative expression of ERP29 and KDM3A was significantly increased when the culture medium was supplemented with EUG 40, indicating a response to ER stress experienced during culture. In conclusion, EUG 40 improved in-vitro follicle survival, activation and development and decreased ROS production, ER stress and histone lysine methylation in goat ovarian tissue.

Modulation of the prostaglandin-endoperoxide synthase 2 gene expression by variant haplotypes: influence of the 3'-untranslated region

The inducible inflammatory enzyme cycloxigenase-2 is up-regulated in cancer, and favors tumor progression. Cycloxigenase-2 is encoded by the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which presents sequence variations in the promoter region (PR) and in the 3′-untranslated region (3′-UTR). Different PR (rs689465, rs689466, rs20417) and 3′-UTR (rs5275) variants were generated by site-directed mutagenesis, and combined in haplotypes to access expression levels using a reporter system (luciferase) in human cells (MCF-7 and HEK293FT). Luciferase activity did not differ significantly among PTGS2 PR constructs, except for pAAC (containing variant allele rs20417 C), with 40% less activity than pAAG (wild-type sequence) in MCF-7 cells (P<0.01). Despite the lack of individual significant differences, PTGS2 PR constructs enclosing rs689466 G (pAGG and pAGC) showed an approximate two-fold increase in luciferase activity when compared to those containing rs689466 A (pAAG, pGAC, pAAC and pGAG) in both cell lines (P<0.001 for MCF-7 and P=0.03 for HEK293FT). The effect of PTGS2 3′-UTR sequences varied between MCF-7 and HEK293FT: MCF-7 cells showed significant reduction (40-60%) in luciferase activity (at least P<0.01), whereas HEK293FT cells showed more diverse results, with an average 2-fold increase when combined constructs (PR and 3′-UTR) were compared to respective parental PR sequences. The contribution of 3′-UTR variant (rs5275) was not consistent in either cell line. Despite the modulation of the 3′-UTR, with variable effects of rs5275, the enhancing transcriptional effect of rs689466 G was still detectable (P<0.0001 in MCF-7 or P=0.03 in HEK293FT cells).

Modulation of the prostaglandin-endoperoxide synthase 2 gene expression by variant haplotypes: influence of the 3'-untranslated region.

The inducible inflammatory enzyme cycloxigenase-2 is up-regulated in cancer, and favors tumor progression. Cycloxigenase-2 is encoded by the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which presents sequence variations in the promoter region (PR) and in the 3'-untranslated region (3'-UTR). Different PR (rs689465, rs689466, rs20417) and 3'-UTR (rs5275) variants were generated by site-directed mutagenesis, and combined in haplotypes to access expression levels using a reporter system (luciferase) in human cells (MCF-7 and HEK293FT). Luciferase activity did not differ significantly among PTGS2 PR constructs, except for pAAC (containing variant allele rs20417 C), with 40% less activity than pAAG (wild-type sequence) in MCF-7 cells (P<0.01). Despite the lack of individual significant differences, PTGS2 PR constructs enclosing rs689466 G (pAGG and pAGC) showed an approximate two-fold increase in luciferase activity when compared to those containing rs689466 A (pAAG, pGAC, pAAC and pGAG) in both cell lines (P<0.001 for MCF-7 and P=0.03 for HEK293FT). The effect of PTGS2 3'-UTR sequences varied between MCF-7 and HEK293FT: MCF-7 cells showed significant reduction (40-60%) in luciferase activity (at least P<0.01), whereas HEK293FT cells showed more diverse results, with an average 2-fold increase when combined constructs (PR and 3'-UTR) were compared to respective parental PR sequences. The contribution of 3'-UTR variant (rs5275) was not consistent in either cell line. Despite the modulation of the 3'-UTR, with variable effects of rs5275, the enhancing transcriptional effect of rs689466 G was still detectable (P<0.0001 in MCF-7 or P=0.03 in HEK293FT cells).

Prototype Development of the Intelligent Hydrogel Wound Dressing and Its Efficacy in the Detection of Model Pathogenic Wound Biofilms.

The early detection of wound infection in situ can dramatically improve patient care pathways and clinical outcomes. There is increasing evidence that within an infected wound the main bacterial mode of living is a biofilm: a confluent community of adherent bacteria encased in an extracellular polymeric matrix. Here we have reported the development of a prototype wound dressing, which switches on a fluorescent color when in contact with pathogenic wound biofilms. The dressing is made of a hydrated agarose film in which the fluorescent dye containing vesicles were mixed with agarose and dispersed within the hydrogel matrix. The static and dynamic models of wound biofilms, from clinical strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis, were established on nanoporous polycarbonate membrane for 24, 48, and 72 h, and the dressing response to the biofilms on the prototype dressing evaluated. The dressing indicated a clear fluorescent/color response within 4 h, only observed when in contact with biofilms produced by a pathogenic strain. The sensitivity of the dressing to biofilms was dependent on the species and strain types of the bacterial pathogens involved, but a relatively higher response was observed in strains considered good biofilm formers. There was a clear difference in the levels of dressing response, when dressings were tested on bacteria grown in biofilm or in planktonic cultures, suggesting that the level of expression of virulence factors is different depending of the growth mode. Colorimetric detection on wound biofilms of prevalent pathogens (S. aureus, P. aeruginosa, and E. faecalis) is also demonstrated using an ex vivo porcine skin model of burn wound infection.

Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model.

The Galleria mellonella infection model was used to assess the in vivo efficacy of phage therapy against laboratory and clinical strains of Pseudomonas aeruginosa. In a first series of experiments, Galleria were infected with the laboratory strain P. aeruginosa PAO1 and were treated with varying multiplicity of infection (MOI) of phages either 2h post-infection (treatment) or 2h pre-infection (prevention) via injection into the haemolymph. To address the kinetics of infection, larvae were bled over a period of 24h for quantification of bacteria and phages. Survival rates at 24h when infected with 10 cells/larvae were greater in the prevention versus treatment model (47% vs. 40%, MOI=10; 47% vs. 20%, MOI=1; and 33% vs. 7%, MOI=0.1). This pattern held true when 100 cells/larvae were used (87% vs. 20%, MOI=10; 53% vs. 13%, MOI=1; 67% vs. 7%, MOI=0.1). By 24h post-infection, phages kept bacterial cell numbers in the haemolymph 1000-fold lower than in the non-treated group. In a second series of experiments using clinical strains to further validate the prevention model, phages protected Galleria when infected with both a bacteraemia (0% vs. 85%) and a cystic fibrosis (80% vs. 100%) isolate. Therefore, this study validates the use of G. mellonella as a simple, robust and cost-effective model for initial in vivo examination of P. aeruginosa-targeted phage therapy, which may be applied to other pathogens with similarly low infective doses.

Combined use of bacteriophage K and a novel bacteriophage to reduce Staphylococcus aureus biofilm formation.

Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-resistant S. aureus (MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a broad host range among S. aureus bacteria. Morphologically, the phage belongs to the Myoviridae family and comprises a large double-stranded DNA (dsDNA) genome of 141,907 bp. DRA88 was mixed with phage K to produce a high-titer mixture that showed strong lytic activity against a wide range of S. aureus isolates, including representatives of the major international MRSA clones and coagulase-negative Staphylococcus. Its efficacy was assessed both in planktonic cultures and when treating established biofilms produced by three different biofilm-producing S. aureus isolates. A significant reduction of biofilm biomass over 48 h of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus biofilms.

Direct detection of a microlens in the Milky Way.

The nature of dark matter remains mysterious, with luminous material accounting for at most approximately 25 per cent of the baryons in the Universe. We accordingly undertook a survey looking for the microlensing of stars in the Large Magellanic Cloud (LMC) to determine the fraction of Galactic dark matter contained in massive compact halo objects (MACHOs). The presence of the dark matter would be revealed by gravitational lensing of the light from an LMC star as the foreground dark matter moves across the line of sight. The duration of the lensing event is the key observable parameter, but gives non-unique solutions when attempting to estimate the mass, distance and transverse velocity of the lens. The survey results to date indicate that between 8 and 50 per cent of the baryonic mass of the Galactic halo is in the form of MACHOs (ref. 3), but removing the degeneracy by identifying a lensing object would tighten the constraints on the mass in MACHOs. Here we report a direct image of a microlens, revealing it to be a nearby low-mass star in the disk of the Milky Way. This is consistent with the expected frequency of nearby stars acting as lenses, and demonstrates a direct determination of a lens mass from a microlensing event. Complete solutions such as this for halo microlensing events will probe directly the nature of the MACHOs.

Community ecology of the metazoan parasites of white croaker, Micropogonias furnieri (Osteichthyes: Sciaenidae), from the coastal zone of the State of Rio de Janeiro, Brazil.

One hundred specimens of white croakers, Micropogonias furnieri (Desmarest 1823) (Osteichthyes: Sciaenidae) collected from Pedra de Guaratiba (23 degrees 01'S, 43 degrees 38'W), State of Rio de Janeiro, Brazil, from September 1997 to August 1999, were necropsied to study their parasites. The majority of the fish (95%) were parasitized by metazoan. Twenty-eight species of parasites were collected. The nematodes were the 40.5% of the total number of parasites specimens collected. Dichelyne elongatus was the most dominant species. Lobatostoma ringens, Pterinotrematoides mexicanum, Corynosoma australe, D. elongatus, and Caligus haemulonis showed a positive correlation between the host's total length and parasite prevalence and abundance. The monogenean P. mexicanum had differences in the prevalence and abundance in relation to sex of the host. The mean diversity in the infracommunities of M. furnieri was H=0.499+/-0.411, with correlation with the host's total length and without differences in relation to sex of the host. One pair of ectoparasites showed positive covariation, and two pairs of endoparasites showed positive association and covariation between their prevalences and abundances, respectively. Negative association or covariations were not found. The dominance of endoparasites in the croakers parasite infracommunities reinforced the differences found in sciaenids from the South American Pacific Ocean, in which the ectoparasites are dominant.