Distribution and Activity of Sulfur-Metabolizing Bacteria along the Temperature Gradient in Phototrophic Mats of the Chilean Hot Spring Porcelana.

In terrestrial hot springs, some members of the microbial mat community utilize sulfur chemical species for reduction and oxidization metabolism. In this study, the diversity and activity of sulfur-metabolizing bacteria were evaluated along a temperature gradient (48-69 °C) in non-acidic phototrophic mats of the Porcelana hot spring (Northern Patagonia, Chile) using complementary meta-omic methodologies and specific amplification of the aprA (APS reductase) and soxB (thiosulfohydrolase) genes. Overall, the key players in sulfur metabolism varied mostly in abundance along the temperature gradient, which is relevant for evaluating the possible implications of microorganisms associated with sulfur cycling under the current global climate change scenario. Our results strongly suggest that sulfate reduction occurs throughout the whole temperature gradient, being supported by different taxa depending on temperature. Assimilative sulfate reduction is the most relevant pathway in terms of taxonomic abundance and activity, whereas the sulfur-oxidizing system (Sox) is likely to be more diverse at low rather than at high temperatures. Members of the phylum Chloroflexota showed higher sulfur cycle-related transcriptional activity at 66 °C, with a potential contribution to sulfate reduction and oxidation to thiosulfate. In contrast, at the lowest temperature (48 °C), Burkholderiales and Acetobacterales (both Pseudomonadota, also known as Proteobacteria) showed a higher contribution to dissimilative sulfate reduction/oxidation as well as to thiosulfate metabolism. Cyanobacteriota and Planctomycetota were especially active in assimilatory sulfate reduction. Analysis of the aprA and soxB genes pointed to members of the order Burkholderiales (Gammaproteobacteria) as the most dominant and active along the temperature gradient for these genes. Changes in the diversity and activity of different sulfur-metabolizing bacteria in photoautotrophic microbial mats along a temperature gradient revealed their important role in hot spring environments, especially the main primary producers (Chloroflexota/Cyanobacteriota) and diazotrophs (Cyanobacteriota), showing that carbon, nitrogen, and sulfur cycles are highly linked in these extreme systems.

Enzyme-Catalyzed Polymerization of Kraft Lignin from Eucalyptus globulus: Comparison of Bacterial and Fungal Laccases Efficacy.

Kraft lignin, a side-stream from the pulp and paper industry, can be modified by laccases for the synthesis of high added-value products. This work aims to study different laccase sources, including a bacterial laccase from Streptomyces ipomoeae (SiLA) and a fungal laccase from Myceliophthora thermophila (MtL), for kraft lignin polymerization. To study the influence of some variables in these processes, a central composite design (CCD) with two continuous variables (enzyme concentration and reaction time) and three levels for each variable was used. The prediction of the behavior of the output variables (phenolic content and molecular weight of lignins) were modelled by means of response surface methodology (RSM). Moreover, characterization of lignins was performed by Fourier-transform infrared (FTIR) spectroscopy and different nuclear magnetic resonance (NMR) spectroscopy techniques. In addition, antioxidant activity was also analyzed. Results showed that lignin polymerization (referring to polymerization as lower phenolic content and higher molecular weight) occurred by the action of both laccases. The enzyme concentration was the most influential variable in the lignin polymerization reaction within the range studied for SiLA laccase, while the most influential variable for MtL laccase was the reaction time. FTIR and NMR characterization analysis corroborated lignin polymerization results obtained from the RSM.

NMR Study on Laccase Polymerization of Kraft Lignin Using Different Enzymes Source.

The usage of laccases is a sustainable and environmentally friendly approach to modifying the Kraft lignin structure for use in certain applications. However, the inherent structure of Kraft lignin, as well as that resulting from laccase modification, still presents challenges for fundamental comprehension and successful lignin valorization. In this study, bacterial and fungal laccases were employed to modify eucalypt Kraft lignin. To evaluate the type and range of the chemical and structural changes of laccase-treated lignins, different NMR techniques, including solution 1H and 2D NMR (heteronuclear single quantum correlation (HSQC)), and solid-state 13C NMR, were applied. Size exclusion chromatography and infrared spectroscopy were also used. Interestingly, HSQC analysis showed substantial changes in the oxygenated aliphatic region of lignins, showing an almost complete absence of signals corresponding to side-chains due to laccase depolymerization. Simultaneously, a significant loss of aromatic signals was observed by HSQC and 1H NMR, which was attributed to a deprotonation of the lignin benzenic rings due to polymerization/condensation by laccase reactions. Then, condensed structures, such as α-5', 5-5', and 4-O-5', were detected by HSQC and 13C NMR, supporting the increment in molecular weight, as well as the phenolic content reduction determined in lignins.

In Vivo Effects of Conditioned Medium from Human Uterine Cervical Stem Cells in an Ovarian Cancer Xenograft Mouse Model.

BACKGROUND/AIM: Ovarian cancer is the most lethal of all gynecological cancers, despite advances in surgical techniques and medical treatments. During the last years, therapies based on mesenchymal stem cells and particularly their secretome (conditioned medium, CM) have emerged as promising treatments for various types of tumors. MATERIALS AND METHODS: In the present study, we evaluated the in vivo antitumor effect of human uterine cervical stem cell conditioned medium (hUCESC-CM) after intraperitoneal administration in an ovarian cancer mouse model. RESULTS: We found that intraperitoneal injection of hUCESC-CM in immunodeficient mice, injected fifty days previously with the human ovarian adenocarcinoma SKOV-3 cell line, significantly reduced abdominal tumor growth, and significantly increased overall survival, compared to control mice. CONCLUSION: hUCESC-CM could be an alternative approach to intraperitoneal treatment of ovarian cancer, either administered alone and/or with conventional chemotherapy.

Extracellular vesicles in mammalian reproduction: a review.

Over the last decades, extracellular vesicles (EVs) have been found to be implicated in a complex universal mechanism of communication between different cell types. EVs are nanostructures of lipid nature that have an exosomal or ectosomal biogenesis, responsible for the intercellular transport of proteins, lipids, carbohydrates, nucleic acids, ions, among other molecules. The content of EVs can vary due to various factors such as hormonal stimuli, non-physiological conditions, metabolic state, etc. Once EVs reach their target cell, they can modulate processes such as gene expression, metabolism, response to external factors, and can even be associated with the delivery of molecules involved in epigenetic inheritance processes in germ cells. In mammalian reproduction, EVs have been shown to play an important role, either in vivo or in vitro, modulating a variety of processes in sperm, oocytes and embryos, and in their respective environments. Moreover, EVs represent a biodegradable, harmless and specific vehicle, which makes them attractive allies to consider when improving assisted reproductive technologies (ARTs). Therefore, the present review aims to describe the content of the main EVs involved in mammalian reproduction and how they can vary due to different factors, as well as to detail how EVs modulate, directly or indirectly, different molecular processes in gametes and embryos. In addition, we will highlight the mechanisms that remain to be elucidated. We will also propose new perspectives according to the characteristics of each particular EV to improve the different ARTs.

Dark Diazotrophy during the Late Summer in Surface Waters of Chile Bay, West Antarctic Peninsula.

Although crucial for the addition of new nitrogen in marine ecosystems, dinitrogen (N2) fixation remains an understudied process, especially under dark conditions and in polar coastal areas, such as the West Antarctic Peninsula (WAP). New measurements of light and dark N2 fixation rates in parallel with carbon (C) fixation rates, as well as analysis of the genetic marker nifH for diazotrophic organisms, were conducted during the late summer in the coastal waters of Chile Bay, South Shetland Islands, WAP. During six late summers (February 2013 to 2019), Chile Bay was characterized by high NO3− concentrations (~20 µM) and an NH4+ content that remained stable near 0.5 µM. The N:P ratio was approximately 14.1, thus close to that of the Redfield ratio (16:1). The presence of Cluster I and Cluster III nifH gene sequences closely related to Alpha-, Delta- and, to a lesser extent, Gammaproteobacteria, suggests that chemosynthetic and heterotrophic bacteria are primarily responsible for N2 fixation in the bay. Photosynthetic carbon assimilation ranged from 51.18 to 1471 nmol C L−1 d−1, while dark chemosynthesis ranged from 9.24 to 805 nmol C L−1 d−1. N2 fixation rates were higher under dark conditions (up to 45.40 nmol N L−1 d−1) than under light conditions (up to 7.70 nmol N L−1 d−1), possibly contributing more than 37% to new nitrogen-based production (≥2.5 g N m−2 y−1). Of all the environmental factors measured, only PO43- exhibited a significant correlation with C and N2 rates, being negatively correlated (p < 0.05) with dark chemosynthesis and N2 fixation under the light condition, revealing the importance of the N:P ratio for these processes in Chile Bay. This significant contribution of N2 fixation expands the ubiquity and biological potential of these marine chemosynthetic diazotrophs. As such, this process should be considered along with the entire N cycle when further reviewing highly productive Antarctic coastal waters and the diazotrophic potential of the global marine ecosystem.

Surface Ammonia-Oxidizer Abundance During the Late Summer in the West Antarctic Coastal System.

Marine ammonia oxidizers that oxidize ammonium to nitrite are abundant in polar waters, especially during the winter in the deeper mixed-layer of West Antarctic Peninsula (WAP) waters. However, the activity and abundance of ammonia-oxidizers during the summer in surface coastal Antarctic waters remain unclear. In this study, the ammonia-oxidation rates, abundance and identity of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were evaluated in the marine surface layer (to 30 m depth) in Chile Bay (Greenwich Island, WAP) over three consecutive late-summer periods (2017, 2018, and 2019). Ammonia-oxidation rates of 68.31 nmol N L-1 day-1 (2018) and 37.28 nmol N L-1 day-1 (2019) were detected from illuminated 2 m seawater incubations. However, high ammonia-oxidation rates between 267.75 and 109.38 nmol N L-1 day-1 were obtained under the dark condition at 30 m in 2018 and 2019, respectively. During the late-summer sampling periods both stratifying and mixing events occurring in the water column over short timescales (February-March). Metagenomic analysis of seven nitrogen cycle modules revealed the presence of ammonia-oxidizers, such as the Archaea Nitrosopumilus and the Bacteria Nitrosomonas and Nitrosospira, with AOA often being more abundant than AOB. However, quantification of specific amoA gene transcripts showed number of AOB being two orders of magnitude higher than AOA, with Nitrosomonas representing the most transcriptionally active AOB in the surface waters. Additionally, Candidatus Nitrosopelagicus and Nitrosopumilus, phylogenetically related to surface members of the NP-ε and NP-γ clades respectively, were the predominant AOA. Our findings expand the known distribution of ammonium-oxidizers to the marine surface layer, exposing their potential ecological role in supporting the marine Antarctic system during the productive summer periods.

Proteorhodopsin Phototrophy in Antarctic Coastal Waters.

Microbial proton-pumping rhodopsins are considered the simplest strategy among phototrophs to conserve energy from light. Proteorhodopsins are the most studied rhodopsins thus far because of their ubiquitous presence in the ocean, except in Antarctica, where they remain understudied. We analyzed proteorhodopsin abundance and transcriptional activity in the Western Antarctic coastal seawaters. Combining quantitative PCR (qPCR) and metagenomics, the relative abundance of proteorhodopsin-bearing bacteria accounted on average for 17, 3.5, and 29.7% of the bacterial community in Chile Bay (South Shetland Islands) during 2014, 2016, and 2017 summer-autumn, respectively. The abundance of proteorhodopsin-bearing bacteria changed in relation to environmental conditions such as chlorophyll a and temperature. Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia were the main bacteria that transcribed the proteorhodopsin gene during day and night. Although green light-absorbing proteorhodopsin genes were more abundant than blue-absorbing ones, the latter were transcribed more intensely, resulting in >50% of the proteorhodopsin transcripts during the day and night. Flavobacteriia were the most abundant proteorhodopsin-bearing bacteria in the metagenomes; however, Alphaproteobacteria and Gammaproteobacteria were more represented in the metatranscriptomes, with qPCR quantification suggesting the dominance of the active SAR11 clade. Our results show that proteorhodopsin-bearing bacteria are prevalent in Antarctic coastal waters in late austral summer and early autumn, and their ecological relevance needs to be elucidated to better understand how sunlight energy is used in this marine ecosystem. IMPORTANCE Proteorhodopsin-bearing microorganisms in the Southern Ocean have been overlooked since their discovery in 2000. The present study identify taxonomy and quantify the relative abundance of proteorhodopsin-bearing bacteria and proteorhodopsin gene transcription in the West Antarctic Peninsula's coastal waters. This information is crucial to understand better how sunlight enters this marine environment through alternative ways unrelated to chlorophyll-based strategies. The relative abundance of proteorhodopsin-bearing bacteria seems to be related to environmental parameters (e.g., chlorophyll a, temperature) that change yearly at the coastal water of the West Antarctic Peninsula during the austral late summers and early autumns. Proteorhodopsin-bearing bacteria from Antarctic coastal waters are potentially able to exploit both the green and blue spectrum of sunlight and are a prevalent group during the summer in this polar environment.

Assessment of Sustainability of Bio Treated Lignocellulose-Based Oleogels.

The development of biological strategies to obtain new high-added value biopolymers from lignocellulosic biomass is a current challenge for scientific community. This study evaluates the biodegradability and ecotoxicity of new formulated oleogels obtained from fermented agricultural residues with Streptomyces, previously reported to show improved rheological and tribological characteristics compared to commercial mineral lubricants. Both new oleogels exhibited higher biodegradation rates than the commercial grease. Classical ecotoxicological bioassays using eukaryotic organisms (Lactuca sativa, Caenorhabditis elegans) showed that the toxic impact of the produced bio-lubricants was almost negligible and comparable to the commercial grease for the target organisms. In addition, high throughput molecular techniques using emerging next-generation DNA-sequencing technologies (NGS) were applied to study the structural changes of lubricant-exposed microbial populations of a standard soil. Results obtained showed that disposal of biomass-based lubricants in the soil environment did not substantially modify the structure and phylogenetic composition of the microbiome. These findings point out the feasibility and sustainability, in terms of biodegradability and eco-safety, of the new bio-lubricants in comparison with commercial mineral greases. This technology entails a promising biological strategy to replace fossil and non-renewable raw materials as well as to obtain useful biopolymers from agricultural residues with potential for large-scale applications.

Upregulation of interferon-alpha gene in bovine embryos produced in vitro in response to experimental infection with noncytophatic bovine-viral-diarrhea virus.

In-vitro fertilization is a routine livestock-breeding technique widely used around the world. Several studies have reported the interaction of bovine viral-diarrhea virus (BVDV) with gametes and in-vitro-produced (IVP) bovine embryos. Since, gene expression in BVDV-infected IVP bovine embryos is scarcely addressed. The aim of this work was to evaluate the differential expression of genes involved in immune and inflammatory response. Groups of 20-25 embryos on Day 6 (morula stage) were exposed (infected) or not (control) to an NCP-BVDV strain in SOF medium. After 24 h, embryos that reached expanded blastocyst stage were washed. Total RNA of each embryo group was extracted to determine the transcription levels of 9 specific transcripts related with antiviral and inflammatory response by SYBR Green real time quantitative (RT-qPCR). Culture media and an aliquot of the last embryos wash on Day 7 were analyzed by titration and virus isolation, respectively. A conventional PCR confirmed BVDV presence in IVP embryos. A significantly higher expression of interferon-α was observed in blastocysts exposed to NCP-BVDV compared to the controls (p < 0.05). In this study, the upregulation of INFα and TLR7 genes involved in inflammatory and immune response in BVDV-infected IVP bovine embryos is a new finding in this field. This differential expression suggest that embryonic cells could function in a manner like immune cells by recognizing and responding early to interaction with viral pathogens. These results provide new insights into the action of BVDV on the complex molecular pathways controlling bovine early embryonic development.

Fischerella thermalis: a model organism to study thermophilic diazotrophy, photosynthesis and multicellularity in cyanobacteria.

The true-branching cyanobacterium Fischerella thermalis (also known as Mastigocladus laminosus) is widely distributed in hot springs around the world. Morphologically, it has been described as early as 1837. However, its taxonomic placement remains controversial. F. thermalis belongs to the same genus as mesophilic Fischerella species but forms a monophyletic clade of thermophilic Fischerella strains and sequences from hot springs. Their recent divergence from freshwater or soil true-branching species and the ongoing process of specialization inside the thermal gradient make them an interesting evolutionary model to study. F. thermalis is one of the most complex prokaryotes. It forms a cellular network in which the main trichome and branches exchange metabolites and regulators via septal junctions. This species can adapt to a variety of environmental conditions, with its photosynthetic apparatus remaining active in a temperature range from 15 to 58 °C. Together with its nitrogen-fixing ability, this allows it to dominate in hot spring microbial mats and contribute significantly to the de novo carbon and nitrogen input. Here, we review the current knowledge on the taxonomy and distribution of F. thermalis, its morphological complexity, and its physiological adaptations to an extreme environment.

Decolorization and detoxification of textile dyes using a versatile Streptomyces laccase-natural mediator system.

Currently, there is increasing interest in assessing the potential of bacterial laccases for industrial and environmental applications especially in harsh conditions. The environmental impact of the textile industry requires novel and effective technologies to mitigate the presence of dyes in wastewaters before discharging into the environment. Dyes usually remain stable in the presence of a variety of chemicals, light and are recalcitrant to microbial degradation. Among available technologies the biological treatments offer environmentally friendly strategies for decolorizing and detoxifying these compounds. The recent discovery of versatile laccases in streptomycetes opens up new opportunities for their commercial application. The aim of this study is to assess the potential of a novel bacterial laccase SilA produced by Streptomyces ipomoeae CECT 3341 active over wide temperature and pH ranges for use as an eco-friendly, biological treatment for the degradation of textile dyes. Insights into the enhancement of the oxidative action of this enzyme through the use of natural redox mediators are presented together with an assessment of the potential toxicity of the degradation products. Our results confirm that the combination of the laccase and natural mediators such as acetosyringone and methyl syringate enhanced the decolorization and detoxification of a variety of textile dyes up to sixfold and 20-fold, respectively. Mediator concentration was found to have a significant effect (p < 0.05) on dye decolorization at 60 °C; thus, the decolorization of Acid Orange 63 increased from 6 to 70-fold when the mediator concentration was increased from 0.1 to 0.5 mM. Further, the toxicity of tartrazine decreased 36-fold when the SilA-MeS system was used to decolorize the dye. The thermal properties of the SilA coupled with the stability of SilA at high pH suggest a potential commercial application for use in the decolorization of textile wastewaters which generally are performed at high temperature (>55 °C) and salinity and neutral pH, conditions which are unfavourable for conventional fungal laccases.

Diurnal Changes in Active Carbon and Nitrogen Pathways Along the Temperature Gradient in Porcelana Hot Spring Microbial Mat.

Composition, carbon and nitrogen uptake, and gene transcription of microbial mat communities in Porcelana neutral hot spring (Northern Chilean Patagonia) were analyzed using metagenomics, metatranscriptomics and isotopically labeled carbon (H13CO3) and nitrogen (15NH4Cl and K15NO3) assimilation rates. The microbial mat community included 31 phyla, of which only Cyanobacteria and Chloroflexi were dominant. At 58°C both phyla co-occurred, with similar contributions in relative abundances in metagenomes and total transcriptional activity. At 66°C, filamentous anoxygenic phototrophic Chloroflexi were >90% responsible for the total transcriptional activity recovered, while Cyanobacteria contributed most metagenomics and metatranscriptomics reads at 48°C. According to such reads, phototrophy was carried out both through oxygenic photosynthesis by Cyanobacteria (mostly Mastigocladus) and anoxygenic phototrophy due mainly to Chloroflexi. Inorganic carbon assimilation through the Calvin-Benson cycle was almost exclusively due to Mastigocladus, which was the main primary producer at lower temperatures. Two other CO2 fixation pathways were active at certain times and temperatures as indicated by transcripts: 3-hydroxypropionate (3-HP) bi-cycle due to Chloroflexi and 3-hydroxypropionate-4-hydroxybutyrate (HH) cycle carried out by Thaumarchaeota. The active transcription of the genes involved in these C-fixation pathways correlated with high in situ determined carbon fixation rates. In situ measurements of ammonia assimilation and nitrogen fixation (exclusively attributed to Cyanobacteria and mostly to Mastigocladus sp.) showed these were the most important nitrogen acquisition pathways at 58 and 48°C. At 66°C ammonia oxidation genes were actively transcribed (mostly due to Thaumarchaeota). Reads indicated that denitrification was present as a nitrogen sink at all temperatures and that dissimilatory nitrate reduction to ammonia (DNRA) contributed very little. The combination of metagenomic and metatranscriptomic analysis with in situ assimilation rates, allowed the reconstruction of day and night carbon and nitrogen assimilation pathways together with the contribution of keystone microorganisms in this natural hot spring microbial mat.

Temperature modulates Fischerella thermalis ecotypes in Porcelana Hot Spring.

In the Porcelana Hot Spring (Northern Patagonia), true-branching cyanobacteria are the dominant primary producers in microbial mats, and they are mainly responsible for carbon and nitrogen fixation. However, little is known about their metabolic and genomic adaptations at high temperatures. Therefore, in this study, a total of 81 Fischerella thermalis strains (also known as Mastigocladus laminosus) were isolated from mat samples in a thermal gradient between 61-46°C. The complementary use of proteomic comparisons from these strains, and comparative genomics of F. thermalis pangenomes, suggested that at least two different ecotypes were present within these populations. MALDI-TOF MS analysis separated the strains into three clusters; two with strains obtained from mats within the upper temperature range (61 and 54°C), and a third obtained from mats within the lower temperature range (51 and 46°C). Both groups possessed different but synonymous nifH alleles. The main proteomic differences were associated with the abundance of photosynthesis-related proteins. Three F. thermalis metagenome assembled genomes (MAGs) were described from 66, 58 and 48°C metagenomes. These pangenomes indicated a divergence of orthologous genes and a high abundance of exclusive genes at 66°C. These results improved the current understanding of thermal adaptation of F. thermalis and the evolution of these thermophilic cyanobacterial species.

Microbial activity during a coastal phytoplankton bloom on the Western Antarctic Peninsula in late summer.

Phytoplankton biomass during the austral summer is influenced by freezing and melting cycles as well as oceanographic processes that enable nutrient redistribution in the West Antarctic Peninsula (WAP). Microbial functional capabilities, metagenomic and metatranscriptomic activities as well as inorganic 13C- and 15N-assimilation rates were studied in the surface waters of Chile Bay during two contrasting summer periods in 2014. Concentrations of Chlorophyll a (Chla) varied from 0.3 mg m-3 in February to a maximum of 2.5 mg m-3 in March, together with a decrease in nutrients; however, nutrients were never depleted. The microbial community composition remained similar throughout both sampling periods; however, microbial abundance and activity changed with Chla levels. An increased biomass of Bacillariophyta, Haptophyceae and Cryptophyceae was observed along with night-grazing activity of Dinophyceae and ciliates (Alveolates). During high Chla conditions, HCO3- uptake rates during daytime incubations increased 5-fold (>2516 nmol C L-1 d-1), and increased photosynthetic transcript numbers that were mainly associated with cryptophytes; meanwhile night time NO3- (>706 nmol N L-1 d-1) and NH4+ (41.7 nmol N L-1 d-1) uptake rates were 2- and 3-fold higher, respectively, due to activity from Alpha-/Gammaproteobacteria and Bacteroidetes (Flavobacteriia). Due to a projected acceleration in climate change in the WAP, this information is valuable for predicting the composition and functional changes in Antarctic microbial communities.

Comparison of the efficiency of bacterial and fungal laccases in delignification and detoxification of steam-pretreated lignocellulosic biomass for bioethanol production.

This study evaluates the potential of a bacterial laccase from Streptomyces ipomoeae (SilA) for delignification and detoxification of steam-exploded wheat straw, in comparison with a commercial fungal laccase from Trametes villosa. When alkali extraction followed by SilA laccase treatment was applied to the water insoluble solids fraction, a slight reduction in lignin content was detected, and after a saccharification step, an increase in both glucose and xylose production (16 and 6%, respectively) was observed. These effects were not produced with T. villosa laccase. Concerning to the fermentation process, the treatment of the steam-exploded whole slurry with both laccases produced a decrease in the phenol content by up to 35 and 71% with bacterial and fungal laccases, respectively. The phenols reduction resulted in an improved performance of Saccharomyces cerevisiae during a simultaneous saccharification and fermentation (SSF) process, improving ethanol production rate. This enhancement was more marked with a presaccharification step prior to the SSF process.

Sperm capacitation pretreatment positively impacts bovine intracytoplasmic sperm injection.

The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is low compared to other species due in part to inadequate egg activation and sperm nucleus decondensation after injection. We hypothesized that this low efficiency is due to the lack of complete sperm capacitation, so we evaluated the effects of isobutylmethylxanthine (IBMX) and methyl-ß-cyclodextrin (MßCD) on bovine sperm capacitation and on the preimplantation developmental potential of bovine embryos generated by ICSI. Treatment with IBMX and MßCD decreased sperm viability (between 13-30%); nevertheless, 0.4 mM IBMX and 1 mM MßCD increased (p < 0.05) capacitation metrics-that is, acrosome exocytosis, intracellular calcium level, plasma membrane fluidity, and tyrosine phosphorylation-compared to the control. After ICSI, embryos injected with IBMX- and MßCD-treated sperm showed similar cleavage to the untreated group (range 82-88%). Pronucleus formation rate was higher with MßCD-pretreatment (54%) compared to the control group (25%), and blastocyst rate was significantly improved with MßCD-pretreatment (24%) compared to the IBMX (18%) and control (17%) groups. Importantly, embryo quality-as assessed by the total number of cells, cell allocation, and apoptotic cell index-was not affected by the sperm treatments. In conclusion, MßCD pretreatment of sperm improved the efficiency of blastocyst production in bovine ICSI.

Improved preimplantation development of bovine ICSI embryos generated with spermatozoa pretreated with membrane-destabilizing agents lysolecithin and Triton X-100.

In cattle, intracytoplasmic sperm injection (ICSI) has a low efficiency. The acrosome content may be responsible for this effect because of the large amount of hydrolytic enzymes that are released within the oocyte. With the aim of removing the acrosome and destabilize the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX) at different concentrations. We evaluated the membrane integrity, the acrosome integrity, DNA integrity, and the variation of phospholipase C zeta. The rates of development (cleavage and blastocysts) were also evaluated along with pronuclear formation and the embryo quality. Spermatozoa incubated with LL and TX (0.01%, 0.02%, 0.03%, and 0.04%) decreased (P < 0.0001) sperm viability in a dose-dependent manner. The acrosome reaction was also increased (P < 0.0001) in all tested concentrations of LL and TX achieving 100% at 0.05% concentration in both treatments. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay reported an increase (P < 0.05) in DNA fragmentation only with the highest concentration of LL (0.06%), whereas all concentrations assessed of TX reported an increased respect to the control. Phospholipase C zeta expression decreased (P < 0.05) in spermatozoa treated with LL and TX at all concentrations tested. A higher cleavage rate was observed in ICSI-TX (66%) and ICSI-LL (65%) groups compared with the untreated control group (51%) and the blastocyst formation rate significantly increased in the ICSI-LL group (29%) compared with the control (21%). No differences were observed in the pronuclear formation and quality of the embryos. In conclusion, the destabilization of the plasma membrane and the release of the acrosomal content with LL and TX before ICSI improve the rate of embryonic development, without affecting the quality of the embryos produced by this technique.

Modulación del estado de óxido-reducción por peróxido de hidrógeno en la etapa de maduración ovocitaria: efecto sobre el desarrollo embrionario en bovinos / Modulation of redox state by hydrogen peroxide in the stage of oocyte maturation: effect on embryonic development in cattle

El estrés oxidativo es definido como un desbalance entre la producción de oxidantes y antioxidantes. La inducción de tolerancia a estrés en los ovocitos conllevaría a un mejor desarrollo embrionario. En bovinos, la incubación de ovocitos maduros con diferentes estresores (térmicos, alta presión hidrostática, oxidativos) incrementaría la tasa de generación de blastocitos. Este estudio evalúa el efecto de la modulación del estado redox incrementando el estrés oxidativo con H2O2 en ovocitos maduros bajo condiciones de cultivo in vitro y su efecto sobre el potencial de desarrollo embrionario. Para ello, ovocitos procedentes de ovarios de matadero fueron madurados en medio TCM-199 suplementado durante 22­23 h, a 38,5 °C, 5 % CO2 y humedad a saturación. Al final de las 22­23 h se incubaron los ovocitos maduros con 0, 50, 100 y 200 µM H2O2. La fecundación in vitro se realizó co-incubando los ovocitos durante 18 h con una concentración final de 1x106 espermatozoides/mL. Los presuntos cigotos fueron denudados y cultivados en medio KSOM-0,4 % BSA a 38,5 °C en atmósfera de baja tensión de O2 (5 % O2, 5 % CO2 y 90 % N2) y humedad a saturación. El estrés oxidativo inducido con H2O2 a una concentración de 50 y 100 µM produce una tasa de división de los embriones similar al control (88,7 %, 83,2 % y 86,4 % respectivamente, p>0,05), disminuyendo significativamente al utilizar una concentración de 200 µM (58,8 %, p<0,05). Asimismo, H2O2 causó un efecto similar en la tasa de blastocitos con 50 µM (20,4 % vs. 25,8 % control, p>0.05) pero disminuyó significativamente con 100 y 200 µM (10,7 % y 3,3 % respectivamente, p<0,05). Es posible, que estos embriones resistentes al estrés oxidativo puedan tener una mayor sobrevida durante los procesos de criopreservación que generan altos niveles de especies reactivas de oxígeno en los embriones.

Oxidative stress is defined as an imbalance between the production of oxidants and antioxidants. The induction of stress tolerance in oocytes leads to a better embryonic development. In cattle incubating mature oocytes with different stressors (thermal, high hydrostatic pressure, oxidative) increase the generation rate of blastocysts. The purpose of this study was to evaluate the effect of modulating the redox state increasing the oxidative stress through H2O2 in mature oocyte under in vitro culture conditions and its effect on the potential of embryonic development. To do this, oocytes from slaughterhouse ovaries were matured in TCM-199 medium supplemented for 22­23 h at 38.5 °C, 5 % CO2 and humidified atmosphere. At the end of 22­23 h, the treatments with 0, 50, 100 and 200 µM H2O2 were applied for 1 h. IVF was performed co-incubating the eggs for 18 h with a final concentration of 1x106 sperm/mL. The presumptive zygotes were denuded and cultured in medium KSOM-0.4 % BSA to 38.5 °C in an atmosphere of low concentration of O2 (5 % O2, 5 % CO2 and 90 % N2) and humidified atmosphere. The results show that the induction of oxidative stress by H2O2 produces a similar effect using a concentration of 50 and 100 mM in the cleavage rate of embryos compared to control (88.7 %, 83.2 % and 86,4 % respectively, p>0.05) and decreasing significantly by using a concentration of 200 mM (58.8 %, p<0.05). Also, H2O2 caused a similar effect on the rate of blastocysts with 50 µM (20.4 % vs. 25.8 control, p>0.05) but decreased significantly with 100 and 200 µM (10.7 % and 3.3 % respectively, p<0.05). It is possible that these embryos resistant to oxidative stress may have a higher survival in the cryopreservation processes that generating high levels of reactive oxygen species.

Influence of process variables on the properties of laccase biobleached pulps.

A laccase stage can be used as a pre-treatment of a standard chemical bleaching sequence to reduce environmental concerns associated to this process. The importance of each independent variable and its influence on the properties of the bleached pulp have been studied in depth in this work, using an adaptive network-based fuzzy inference system (ANFIS) with four independent variables (laccase, buffer, mediator and oxygen) as input. Eucalyptus globulus kraft pulp was biobleached using a laccase from Pycnoporus sanguineus and a natural mediator (acetosyringone). Later, an alkaline extraction and a hydrogen peroxide treatment were applied. Most biobleaching processes showed a decrease in kappa number and an increase in brightness with no significant impact on the viscosity values, compared with the control. Oxygen was the variable with the smallest influence on the final pulp properties while the laccase and buffer solution showed a significant influence.