Effect of Aphidicolin, a Reversible Inhibitor of Eukaryotic Nuclear DNA Replication, on the Production of Genetically Modified Porcine Embryos by CRISPR/Cas9.

Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 µM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.

Effects of pomegranate peel extract on ruminal and post-ruminal in vitro degradation of rumen inoculum of the dairy cow.

This experiment was carried out to study the effect of water extracted pomegranate peel extract (PE) on ruminal protein degradation and post-ruminal digestion in the dairy cow. PE was added at six levels of total phenolics (g/kg of the basal diet); 3.75 (PE1); 4.4 (PE2); 5.05 (PE3); 5.70 (PE4); and 6.35 (PE5). Rumen degradable crude protein (rdCP) decreased with PE addition (L < 0.0001), but total CP degradability (tdCP) was not affected. Compared to PE0, PE2, and PE3 diets showed higher (L = 0.054, Q = 0.029) digestibility of bypass CP (dBCP). Increasing levels of PE resulted in a decrease in proteolytic bacteria numbers (p < 0.0001). At PE4 and PE5 levels, total VFA and acetate concentrations linearly decreased compared to PE0. PE inclusion lowered the acetate:propionate ratio (L = 0.0001) and Ammonia-N production after 24 h (L = 0.0008) of incubation. The total number of protozoa, genera Dasytricha and Isotricha, and subfamilies Entodiniinae, Diplodiniinae, and Ophrioscolecinae decreased with increasing dietary PE concentration (p < 0.0001). The results suggest that all levels of PE addition reduce the protozoal population and Ammonia-N concentration. All PE levels slowed down protein degradation in the rumen but PE2 and PE3 showed the greatest effect.

Green biosynthesis of silver nanoparticles by Aspergillus niger and its antiamoebic effect against Allovahlkampfia spelaea trophozoite and cyst.

BACKGROUND: Fungi represent an interesting candidate for the synthesis of nanoparticles. The biosynthesis of silver nanoparticles (AgNPs) has many industrial and biomedical indications. We aimed in this work to biologically synthesize silver nanoparticles using Aspergillus niger and to evaluate its effect against the newly identified Allovahlkampfia spelaea that causes resistant human keratitis. MATERIAL AND METHODS: Aspergillus niger (soil isolate) was treated with silver nitrate to produce silver nanoparticles. AgNPs were characterized by Ultraviolet-Visible Spectroscopy, Transmission Electron Microscopy, and Fourier Transform Infrared Spectroscopy. The effect of the synthesized nanoparticles against Allovahlkampfia spelaea growth, encystation, excystation, and toxicity in host cells was evaluated. RESULTS: AgNPs exhibited significant inhibition of Allovahlkampfia spelaea viability and growth of both trophozoites and cysts, with a reduction of amoebic cytotoxic activity in host cells. CONCLUSION: AgNPs may give a promising future to the treatment of Allovahlkampfia spelaea infections in humans.

Aminoglycoside interactions and impacts on the eukaryotic ribosome.

Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages (initiation, elongation, termination, and recycling) of the translation mechanism. The propensity of aminoglycosides to induce miscoding errors that suppress the termination of protein synthesis supports their potential as therapeutic interventions in human diseases associated with premature termination codons (PTCs). However, the sites of interaction of aminoglycosides with the eukaryotic ribosome and their modes of action in eukaryotic translation remain largely unexplored. Here, we use the combination of X-ray crystallography and single-molecule FRET analysis to reveal the interactions of distinct classes of aminoglycosides with the 80S eukaryotic ribosome. Crystal structures of the 80S ribosome in complex with paromomycin, geneticin (G418), gentamicin, and TC007, solved at 3.3- to 3.7-Å resolution, reveal multiple aminoglycoside-binding sites within the large and small subunits, wherein the 6'-hydroxyl substituent in ring I serves as a key determinant of binding to the canonical eukaryotic ribosomal decoding center. Multivalent binding interactions with the human ribosome are also evidenced through their capacity to affect large-scale conformational dynamics within the pretranslocation complex that contribute to multiple aspects of the translation mechanism. The distinct impacts of the aminoglycosides examined suggest that their chemical composition and distinct modes of interaction with the ribosome influence PTC read-through efficiency. These findings provide structural and functional insights into aminoglycoside-induced impacts on the eukaryotic ribosome and implicate pleiotropic mechanisms of action beyond decoding.

Short-term impact of biochar amendments on eukaryotic communities in three different soils.

This study determined the loading impacts of wood-based biochar on the eukaryotic community in three different soils (brown sandy loam-BSL, red loam-RL and a black clay loam-BCL) using a pot trial conducted over 10 months. Soil analysis and 18S rRNA gene sequencing performed using the Illumina MiSeq platform was carried out to evaluate the changes in eukaryotic community composition in relation to different added amounts of biochar. It was found that biochar addition had a negligible effect on diversity parameters in the brown sandy loam Kurosol (BSL) and red loam Dermosol (RL) soils. There were, however, significant changes in eukaryotic community composition of these biochar amended soils. These changes were most discernible in the lighter (low clay content) BSL soil for the fungal communities (F = 3.0106, p = 0.0003) present and also when total eukaryotes were considered (F = 2.3907, p = 0.0002). In this respect Glomeromycota seem to be slightly promoted in the lighter BSL soils, which might be due to increased soil porosity and soil chemical fertility. Clay rich BCL soil community structure correlated to a greater degree with soil chemistry influenced by biochar addition. The results showed that soil microeukaryotes were affected by short term carbon amendment, though to a limited extent. The limited effect of biochar loading rates on the soil microbiology could be due to the short incubation period, the lack of added fertiliser nutrients, and also the inherent stability of the soil eukaryotic community. The data suggested the impacts that were observed however included important plant symbiotic organisms. The results also imply biochar applications at different loading levels have differential effects on soil microeurokaryotes in relation to soil properties in particular clay content.

Natural mechanisms and artificial PEG-induced mechanism that repair traumatic damage to the plasmalemma in eukaryotes.

Eukaryotic tissues are composed of individual cells surrounded by a plasmalemma that consists of a phospholipid bilayer with hydrophobic heads that bind cell water. Bound-water creates a thermodynamic barrier that impedes the fusion of a plasmalemma with other membrane-bound intracellular structures or with the plasmalemma of adjacent cells. Plasmalemmal damage consisting of small or large holes or complete transections of a cell or axon results in calcium influx at the lesion site. Calcium activates fusogenic pathways that have been phylogenetically conserved and that lower thermodynamic barriers for fusion of membrane-bound structures. Calcium influx also activates phylogenetically conserved sealing mechanisms that mobilize the gradual accumulation and fusion of vesicles/membrane-bound structures that seal the damaged membrane. These naturally occurring sealing mechanisms for different cells vary based on the type of lesion, the type of cell, the proximity of intracellular membranous structures to the lesion and the relation to adjacent cells. The reliability of different measures to assess plasmalemmal sealing need be carefully considered for each cell type. Polyethylene glycol (PEG) bypasses calcium and naturally occurring fusogenic pathways to artificially fuse adjacent cells (PEG-fusion) or artificially seal transected axons (PEG-sealing). PEG-fusion techniques can also be used to rapidly rejoin the closely apposed, open ends of severed axons. PEG-fused axons do not (Wallerian) degenerate and PEG-fused nerve allografts are not immune-rejected, and enable behavioral recoveries not observed for any other clinical treatment. A better understanding of natural and artificial mechanisms that induce membrane fusion should provide better clinical treatment for many disorders involving plasmalemmal damage.

Parasitic Protozoa: Unusual Roles for G-Quadruplexes in Early-Diverging Eukaryotes.

Guanine-quadruplex (G4) motifs, at both the DNA and RNA levels, have assumed an important place in our understanding of the biology of eukaryotes, bacteria and viruses. However, it is generally little known that their very first description, as well as the foundational work on G4s, was performed on protozoans: unicellular life forms that are often parasitic. In this review, we provide a historical perspective on the discovery of G4s, intertwined with their biological significance across the protozoan kingdom. This is a history in three parts: first, a period of discovery including the first characterisation of a G4 motif at the DNA level in ciliates (environmental protozoa); second, a period less dense in publications concerning protozoa, during which DNA G4s were discovered in both humans and viruses; and third, a period of renewed interest in protozoa, including more mechanistic work in ciliates but also in pathogenic protozoa. This last period has opened an exciting prospect of finding new anti-parasitic drugs to interfere with parasite biology, thus adding new compounds to the therapeutic arsenal.

Canonical translation-modulating OFF-riboswitches with a single aptamer binding to a small molecule that function in a higher eukaryotic cell-free expression system.

We have found that OFF-riboswitches that ligand-dependently downregulate the canonical translation in a higher eukaryotic expression system (wheat germ extract) can be easily created by inserting a single aptamer into the 5' untranslated region (UTR) of mRNA, even if its ligand is as small as theophylline. The key is the position of the inserted aptamer: the 5' end (+0 position) is much better than other positions for inhibiting canonical translation with the aptamer-ligand complex. The data showed that ribosome loading is suppressed by a rigid structure in the 5' end, and this suppression is dependent on the structure's stability but not on its size. Although this preference of aptamer insertion point contradicts the results in a lower eukaryote, it accords with the fact that the 5'-end structural hindrance is more effective for blocking the ribosome in higher eukaryotes. Therefore, the present type of OFF-riboswitch would function in various higher eukaryotic expression systems.

Identification of the streptothricin and tunicamycin biosynthetic gene clusters by genome mining in Streptomyces sp. strain fd1-xmd.

The genus Streptomyces have been highly regarded for their important source of natural products. Combined with the technology of genome sequencing and mining, we could identify the active ingredients from fermentation broth quickly. Here, we report on Streptomyces sp. strain fd1-xmd, which was isolated from a soil sample collected in Shanghai. Interestingly, the fermentation broth derived from this strain demonstrated broad-spectrum antimicrobial activity against gram-positive bacteria, gram-negative bacteria, and eukaryotes. To identify the antimicrobial substances and their biosynthetic gene clusters, we sequenced the fd1-xmd strain and obtained a genome 7,929,999 bp in length. The average GC content of the chromosome was 72.5 mol%. Knockout experiments demonstrated that out of eight biosynthetic gene clusters we could identify, two are responsible for the biosynthesis of the antibiotics streptothricin (ST) and tunicamycin (TM). The ST biosynthetic gene cluster from fd1-xmd was verified via successful heterologous expression in Streptomyces coelicolor M1146. ST production had a yield of up to 0.5 g/L after the optimization of culture conditions. This study describes a novel producer of ST and TM and outlines the complete process undertaken for Streptomyces sp. strain fd1-xmd genome mining.

Can heavy isotopes increase lifespan? Studies of relative abundance in various organisms reveal chemical perspectives on aging.

Stable heavy isotopes co-exist with their lighter counterparts in all elements commonly found in biology. These heavy isotopes represent a low natural abundance in isotopic composition but impose great retardation effects in chemical reactions because of kinetic isotopic effects (KIEs). Previous isotope analyses have recorded pervasive enrichment or depletion of heavy isotopes in various organisms, strongly supporting the capability of biological systems to distinguish different isotopes. This capability has recently been found to lead to general decline of heavy isotopes in metabolites during yeast aging. Conversely, supplementing heavy isotopes in growth medium promotes longevity. Whether this observation prevails in other organisms is not known, but it potentially bears promise in promoting human longevity.

Ammonium removal using algae-bacteria consortia: the effect of ammonium concentration, algae biomass, and light.

In this study, the effects of ammonium nitrogen concentration, algae biomass concentration, and light conditions (wavelength and intensity) on the ammonium removal efficiency of algae-bacteria consortia from wastewater were investigated. The results indicated that ammonium concentration and light intensity had a significant impact on nitrification. It was found that the highest ammonia concentration (430 mg N/L) in the influent resulted in the highest ammonia removal rate of 108 ± 3.6 mg N/L/days, which was two times higher than the influent with low ammonia concentration (40 mg N/L). At the lowest light intensity of 1000 Lux, algae biomass concentration, light wavelength, and light cycle did not show a significant effect on the performance of algal-bacterial consortium. Furthermore, the ammonia removal rate was approximately 83 ± 1.0 mg N/L/days, which was up to 40% faster than at the light intensity of 2500 Lux. It was concluded that the algae-bacteria consortia can effectively remove nitrogen from wastewater and the removal performance can be stabilized and enhanced using the low light intensity of 1000 Lux that is also a cost-effective strategy.

Effects of tetracycline and ibuprofen on the relative abundance of microbial eukaryotic and bacterial populations in wastewater treatment.

The activated sludge process in a wastewater treatment plant (WWTP) relies on the activity of microbes to reduce the organic and inorganic matter and produce effluent that is safe to discharge into receiving waters. This research examined the effects of the non-steroidal anti-inflammatory drug ibuprofen and the antibiotic tetracycline on the relative abundance and composition of eukaryotes and bacteria in the microbial population present in activated sludge from a WWTP. The current investigation was designed to observe the impact of these contaminants, at low (environmentally relevant concentrations) as well as high concentrations of the drugs. Using 16S and 18S rRNA gene primer sets and quantitative polymerase chain reaction, the abundance of each population was monitored as well as the relative ratio of the two populations under the various conditions. It was found that current environmentally relevant concentrations of ibuprofen (100 ng/mL) stimulated eukaryotic growth but higher concentrations (2,000 ng/mL, 100,000 ng/mL) reduced their numbers significantly especially in the presence of tetracycline. Finally using denaturing gradient gel electrophoresis, some of the more abundant eukaryotes were identified and it was noted that high ibuprofen and tetracycline concentrations favoured the abundance of some genera.

Interspecific variability in phosphorus-induced lipid remodelling among marine eukaryotic phytoplankton.

The response of marine microalgal lipids to phosphorus is of central importance in phytoplankton ecology but remains poorly understood. We determined how taxonomically diverse microalgal species remodelled their lipid class profile in response to phosphorus availability and whether these changes coincided with those already known to occur in land plants and in the limited number of phytoplankton species for which data are available. The complete lipid class profile and specific lipid ratios influenced by phosphorus availability were quantified in two green microalgae and seven Chromalveolates exposed to phosphorus repletion, deprivation and replenishment. Lipid class cell quota changes in the two green microalgae resembled the currently described pattern of betaine lipids substituting for phospholipids under phosphorus depletion, whereas only two of the studied Chromalveolates showed this pattern. Sulpholipids counterbalanced phosphatidylglycerol only in Picochlorum atomus. In all other species, both lipids decreased simultaneously under phosphorus deprivation, although sulpholipids declined more slowly. Phosphorus deprivation always induced a decrease in digalactosyl-diacylglycerol. However, the ratio of digalactosyl-diacylglycerol to total phospholipids increased in eight species and remained unchanged in Isochrysis galbana. Marine phytoplankton seems to have evolved a diversified mechanism for remodelling its lipid class profile under the influence of phosphorus, with cryptophytes and particularly haptophytes exhibiting previously unobserved lipid responses to phosphorus.

Alkamides from Anacyclus pyrethrum L. and Their in Vitro Antiprotozoal Activity.

In our ongoing study to evaluate the antiprotozoal activity of alkamides from Asteraceae, a dichloromethane extract from the roots of Anacycluspyrethrum L. showed a moderate in vitro activity against the NF54 strain of Plasmodium falciparum and against Leishmaniadonovani (amastigotes, MHOM/ET/67/L82 strain). Seven pure alkamides and a mixture of two further alkamides were isolated by column chromatography followed by preparative high performance liquid chromatography. The alkamides were identified by mass- and NMR-spectroscopic methods as tetradeca-2E,4E-dien-8,10-diynoic acid isobutylamide (anacycline, 1), deca-2E,4E-dienoic acid isobutylamide (pellitorine, 2), deca-2E,4E,9-trienoic acid isobutylamide (3), deca-2E,4E-dienoic acid 2-phenylethylamide (4), undeca-2E,4E-dien-8,10-diynoic acid isopentylamide (5), tetradeca-2E,4E,12Z-trien-8,10-diynoic acid isobutylamide (6), and dodeca-2E,4E-dien acid 4-hydroxy-2-phenylethylamide (7). Two compounds-undeca-2E,4E-dien-8,10-diynoic acid 2-phenylethylamide (8) and deca-2E,4E-dienoic acid 4-hydroxy-2-phenylethylamide (9)-were isolated as an inseparable mixture (1:4). Compounds 3, 4, and 5 were isolated from Anacycluspyrethrum L. for the first time. While compounds 4 and 5 were previously known from the genus Achillea, compound 3 is a new natural product, to the best of our knowledge. All isolated alkamides were tested in vitro for antiprotozoal activity against Plasmodium falciparum, Trypanosomabruceirhodesiense, Trypanosomacruzi, and Leishmaniadonovani and for cytotoxicity against L6 rat skeletal myoblasts.

Pesticide seed dressings can affect the activity of various soil organisms and reduce decomposition of plant material.

BACKGROUND: Seed dressing with pesticides is widely used to protect crop seeds from pest insects and fungal diseases. While there is mounting evidence that especially neonicotinoid seed dressings detrimentally affect insect pollinators, surprisingly little is known on potential side effects on soil biota. We hypothesized that soil organisms would be particularly susceptible to pesticide seed dressings as they get in direct contact with these chemicals. Using microcosms with field soil we investigated, whether seeds treated either with neonicotinoid insecticides or fungicides influence the activity and interaction of earthworms, collembola, protozoa and microorganisms. The full-factorial design consisted of the factor Seed dressing (control vs. insecticide vs. fungicide), Earthworm (no earthworms vs. addition Lumbricus terrestris L.) and collembola (no collembola vs. addition Sinella curviseta Brook). We used commercially available wheat seed material (Triticum aesticum L. cf. Lukullus) at a recommended seeding density of 367 m(-2). RESULTS: Seed dressings (particularly fungicides) increased collembola surface activity, increased the number of protozoa and reduced plant decomposition rate but did not affect earthworm activity. Seed dressings had no influence on wheat growth. Earthworms interactively affected the influence of seed dressings on collembola activity, whereas collembola increased earthworm surface activity but reduced soil basal respiration. Earthworms also decreased wheat growth, reduced soil basal respiration and microbial biomass but increased soil water content and electrical conductivity. CONCLUSIONS: The reported non-target effects of seed dressings and their interactions with soil organisms are remarkable because they were observed after a one-time application of only 18 pesticide treated seeds per experimental pot. Because of the increasing use of seed dressing in agriculture and the fundamental role of soil organisms in agroecosystems these ecological interactions should receive more attention.

David and Goliath: chemical perturbation of eukaryotes by bacteria.

Environmental microbes produce biologically active small molecules that have been mined extensively as antibiotics and a smaller number of drugs that act on eukaryotic cells. It is known that there are additional bioactives to be discovered from this source. While the discovery of new antibiotics is challenged by the frequent discovery of known compounds, we contend that the eukaryote-active compounds may be less saturated. Indeed, despite there being far fewer eukaryotic-active natural products these molecules interact with a far richer diversity of molecular and cellular targets.

INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(Phenyl) Tetrazolium Chloride) Is Toxic to Prokaryote Cells Precluding Its Use with Whole Cells as a Proxy for In Vivo Respiration.

Prokaryote respiration is expected to be responsible for more than half of the community respiration in the ocean, but the lack of a practical method to measure the rate of prokaryote respiration in the open ocean resulted in very few published data leaving the role of organotrophic prokaryotes open to debate. Oxygen consumption rates of oceanic prokaryotes measured with current methods may be biased due to pre-incubation size filtration and long incubation times both of which can change the physiological and taxonomic profile of the sample during the incubation period. In vivo INT reduction has been used in terrestrial samples to estimate respiration rates, and recently, the method was introduced and applied in aquatic ecology. We measured oxygen consumption rates and in vivo INT reduction to formazan in cultures of marine bacterioplankton communities, Vibrio harveyi and the eukaryote Isochrysis galbana. For prokaryotes, we observed a decrease in oxygen consumption rates with increasing INT concentrations between 0.05 and 1 mM. Time series after 0.5 mM INT addition to prokaryote samples showed a burst of in vivo INT reduction to formazan and a rapid decline of oxygen consumption rates to zero within less than an hour. Our data for non-axenic eukaryote cultures suggest poisoning of the eukaryote. Prokaryotes are clearly poisoned by INT on time scales of less than 1 h, invalidating the interpretation of in vivo INT reduction to formazan as a proxy for oxygen consumption rates.

Comparative metatranscriptomics identifies molecular bases for the physiological responses of phytoplankton to varying iron availability.

In vast expanses of the oceans, growth of large phytoplankton such as diatoms is limited by iron availability. Diatoms respond almost immediately to the delivery of iron and rapidly compose the majority of phytoplankton biomass. The molecular bases underlying the subsistence of diatoms in iron-poor waters and the plankton community dynamics that follow iron resupply remain largely unknown. Here we use comparative metatranscriptomics to identify changes in gene expression associated with iron-stimulated growth of diatoms and other eukaryotic plankton. A microcosm iron-enrichment experiment using mixed-layer waters from the northeastern Pacific Ocean resulted in increased proportions of diatom transcripts and reduced proportions of transcripts from most other taxa within 98 h after iron addition. Hundreds of diatom genes were differentially expressed in the iron-enriched community compared with the iron-limited community; transcripts of diatom genes required for synthesis of photosynthesis and chlorophyll components, nitrate assimilation and the urea cycle, and synthesis of carbohydrate storage compounds were significantly overrepresented. Transcripts of genes encoding rhodopsins in eukaryotic phytoplankton were significantly underrepresented following iron enrichment, suggesting rhodopsins help cells cope with low-iron conditions. Oceanic diatoms appear to display a distinctive transcriptional response to iron enrichment that allows chemical reduction of available nitrogen and carbon sources along with a continued dependence on iron-free photosynthetic proteins rather than substituting for iron-containing functional equivalents present within their gene repertoire. This ability of diatoms to divert their newly acquired iron toward nitrate assimilation may underlie why diatoms consistently dominate iron enrichments in high-nitrate, low-chlorophyll regions.

Understanding drug resistance in human intestinal protozoa.

Infections with intestinal protozoa continue to be a major health problem in many areas of the world. The widespread use of a limited number of therapeutic agents for their management and control raises concerns about development of drug resistance. Generally, the use of any antimicrobial agent should be accompanied by meticulous monitoring of its efficacy and measures to minimize resistance formation. Evidence for the occurrence of drug resistance in different intestinal protozoa comes from case studies and clinical trials, sometimes with a limited number of patients. Large-scale field-based assessment of drug resistance and drug sensitivity testing of clinical isolates are needed. Furthermore, the association of drug resistance with certain geographic isolates or genotypes deserves consideration. Drug resistance has been triggered in vitro and has been linked to modification of pyruvate:ferredoxin oxidoreductase, nitroreductases, antioxidant defense, or cytoskeletal system. Further mechanistic studies will have important implications in the development of second generation therapeutic agents.

Osmosensing and osmoregulation in unicellular eukaryotes.

Eukaryotic microorganisms possess mechanisms to detect osmotic variations in their surroundings, from specialized receptors and membrane transporters, to sophisticated systems such as two-component histidine kinases. Osmotic stimuli are transduced through conserved phosphorylation cascades that result in a rapid response to mitigate stress. This response allows for the maintenance of an optimal biochemical environment for cell functioning, as well as a suitable recovery in suboptimal environments that would otherwise endanger cell survival. The molecular basis of these responses has been largely studied in yeasts and bacteria. However, fewer studies have been published concerning the molecular basis of osmoregulation in other eukaryotic microorganisms such as protozoans and microalgae. Here, we review the main osmosensors reported in unicellular eukaryotic microorganisms (yeasts, microalgae and protozoa) and the pathways that maintain homeostasis in cells encountering hyperosmotic challenges.