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At least 19 records

Ciliated protozoa of two antarctic lakes: analysis by quantitative protargol staining and examination of artificial substrates

Planktonic and artificial substrate-associated ciliates have been identified in two perennially ice-covered antarctic lakes of the McMurdo Dry Valleys. Abundances estimated by quantitative protargol staining ranged from < 5 to 31690 cells l-1, levels that are comparable to those previously obtained using other methods. Nineteen ciliate taxa were identified from these lakes, with the most frequently encountered genera being Plagiocampa, Askenasia, Monodinium, Sphaerophrya and Vorticella. The taxonomic findings compare favorably with those of previous investigators; however four previously unreported genera were observed in both Lakes Fryxell and Hoare. The variability in the depth distributions of ciliates in Lake Fryxell is explained in terms of lake physicochemical properties and ciliate prey distributions, while factors related to temporal succession in the Lake Hoare assemblage remain unexplained. Local marine or temperate zone freshwater habitats are a more likely source than the surrounding dry valleys soils for present ciliate colonists in these lakes. Although the taxonomic uncertainties require further examination, our results suggest that ciliate populations in these antarctic lakes undergo significant fluctuations and are more diverse than was previously recognized.

NASA Discipline Exobiology↗

Role of interferon in resistance and immunity to protozoa

Production of interferon (I) in response to protozoan infection, and the interferon-mediated inhibition of parasite replication were studied in order to determine if these effects may be related to immunologic-mediated resistance of the hosts. Two extracellular parasites-Trypanosoma brucei rhodesiense and Naegleria fowlei were used. Upon infection with the trypanosome, only resistant strains of mice produced I. An early peak of alpha/beta I is followed by appearance of gamma I, which coincided with antibody production and a drop in parasitemia. In case of the amoeba, pretreatment of its suspension with alpha/beta I inhibits its replication in vitro, and appears to protect mice from the infection and the disease. It is proposed that production of interferon, with its regulatory effect on the immune responses, may play a major role in regulating the processes of protozoan-caused diseases.

Sonnenfeld, G.↗

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa, and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 h in nylon bags within the rumen of cannulated dairy cows. Across a gene catalog covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of hemicelluloses, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8, and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggest that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

Hagen, Live↗

A biosensor for cadmium based on bioconvective patterns

An 'in vitro' method for monitoring cadmium, one of the most lethal bivalent heavy metals, can detect biologically active levels. The effects of cadmium tend to concentrate in protozoa far above natural levels and therein begin transferring through freshwater food chains to animals and humans. In a small sample volume (approximately 5 ml) the method uses the toxic response to the protozoa, Tetrahymena pyriformis, to cadmium. The assay relies on macroscopic bioconvective patterns to measure the toxic response, giving a sensitivity better than 1 micro-g/1 and a toxicity threshold to 7 micro-g/1 for Cd(2+). Cadmium hinders pattern formation in a dose-dependent manner. Arrested organism growth arises from slowed division and mutation to non-dividing classes. Unlike previous efforts, this method can be performed in a shallow flow device and does not require electronic or chemical analyses to monitor toxicity.

Noever, David A.↗

Microbial Diffraction Gratings as Optical Detectors for Heavy Metal Pollutants

As a significant industrial pollutant, cadmium is implicated as the cause of itai-itai disease. For biological detection of cadmium toxicity, an assay device has been developed using the motile response of the protozoa species, Tetrahymena pyriformis. This mobile protozoa measures 50 microns in diameter, swims at 10 body lengths per second, and aggregates into macroscopically visible patterns at high organism concentrations. The assay demonstrates a Cd(+2) sensitivity better than 1 micro-M and a toxicity threshold to 5 micro-M, thus encouraging the study of these microbial cultures as viable pollution detectors. Using two-dimensional diffraction patterns within a Tetrahymena culture, the scattered light intensity varies with different organism densities (population counts). The resulting density profile correlates strongly with the toxic effects at very low dosages for cadmium (less than 5 ppm) and then for poison protection directly (with nickel and copper antagonists competing with cadmium absorption). In particular, copper dosages as low as 0.1-0.5 mM Cu have shown protective antagonism against cadmium, have enhanced density variability for cultures containing 1 mM Cd(+2) and therefore have demonstrated the sensitivity of the optical detection system. In this way, such microbial diffraction patterns give a responsive optical measure of biological culture changes and toxicity determination in aqueous samples of heavy metals and industrial pollutants.

Noever, David↗

Molecular indicators for palaeoenvironmental change in a Messinian evaporitic sequence (Vena del Gesso, Italy). II: High-resolution variations in abundances and 13C contents of free and sulphur-bound carbon skeletons in a single marl bed

The extractable organic matter of 10 immature samples from a marl bed of one evaporitic cycle of the Vena del Gesso sediments (Gessoso-solfifera Fm., Messinian, Italy) was analyzed quantitatively for free hydrocarbons and organic sulphur compounds. Nickel boride was used as a desulphurizing agent to recover sulphur-bound lipids from the polar and asphaltene fractions. Carbon isotopic compositions (delta vs PDB) of free hydrocarbons and of S-bound hydrocarbons were also measured. Relationships between these carbon skeletons, precursor biolipids, and the organisms producing them could then be examined. Concentrations of S-bound lipids and free hydrocarbons and their delta values were plotted vs depth in the marl bed and the profiles were interpreted in terms of variations in source organisms, 13 C contents of the carbon source, and environmentally induced changes in isotopic fractionation. The overall range of delta values measured was 24.7%, from -11.6% for a component derived from green sulphur bacteria (Chlorobiaceae) to -36.3% for a lipid derived from purple sulphur bacteria (Chromatiaceae). Deconvolution of mixtures of components deriving from multiple sources (green and purple sulphur bacteria, coccolithophorids, microalgae and higher plants) was sometimes possible because both quantitative and isotopic data were available and because either the free or S-bound pool sometimes appeared to contain material from a single source. Several free n-alkanes and S-bound lipids appeared to be specific products of upper-water-column primary producers (i.e. algae and cyanobacteria). Others derived from anaerobic photoautotrophs and from heterotrophic protozoa (ciliates), which apparently fed partly on Chlorobiaceae. Four groups of n-alkanes produced by algae or cyanobacteria were also recognized based on systematic variations of abundance and isotopic composition with depth. For hydrocarbons probably derived from microalgae, isotopic variations are well correlated with those of total organic carbon. A resistant aliphatic biomacromolecule produced by microalgae is, therefore, probably an important component of the kerogen. These variations reflect changes in the depositional environment and early diagenetic transformations. Changes in the concentrations of S-bound lipids induced by variations in conditions favourable for sulphurization were discriminated from those related to variations in primary producer assemblages. The water column of the lagoonal basin was stratified and photic zone anoxia occurred during the early and middle stages of marl deposition. During the last stage of the marl deposition the stratification collapsed due to a significant shallowing of the water column. Contributions from anaerobic photoautotrophs were apparently associated with variations in depth of the chemocline.

Non-NASA Center↗

The crystal structure of glucokinase from Leishmania braziliensis

Glucokinase from pathogenic protozoa of the genus Leishmania is a potential drug target for the chemotherapeutic treatment against leishmaniasis because this enzyme is located at a nodal point between two critically important metabolic pathways, glycolysis and the pentose phosphate pathway (PPP). L. braziliensis glucokinase (LbGlcK) was evaluated for its structural characterization and enzymatic performance. The enzyme catalyzes the phosphorylation of d-glucose with co-substrate ATP to yield the products G6P and ADP. LbGlcK had K M values determined as 6.61 ± 2.63 mM and 0.338 ± 0.080 mM for d-glucose and ATP, respectively. The 1.85 Å resolution X-ray crystal structure of the apo form of LbGlcK was determined and a homodimer was revealed where each subunit (both in open conformations) included the typical small and large domains. Structural comparisons were assessed in relationship to Homo sapiens hexokinase IV and Trypanosoma cruzi glucokinase. Comparisons revealed that all residues important for making hydrogen bonding interactions with d-glucose in the active site and catalysis were strictly conserved. LbGlcK was screened against four glucosamine analogue inhibitors and the stronger inhibitor of the series, HPOP-GlcN, had a K i value of 56.9 ± 16.6 μM that exhibited competitive inhibition. Finally, for the purpose of future structure-based drug design experimentation, L. braziliensis glucokinase was observed to be very similar to T. cruzi glucokinase even though there was a 44% protein sequence identity between the two enzymes.

59 BASIC BIOLOGICAL SCIENCES↗

Identification of Potent and Selective Inhibitors of Acanthamoeba : Structural Insights into Sterol 14α-Demethylase as a Key Drug Target

Acanthamoeba are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. Acanthamoeba sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC 50 0.45 μM), VT1598 (0.25 μM), and VT1161 (0.20 μM). The crystal structures of A. castellanii CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.

60 APPLIED LIFE SCIENCES↗

Passively Sampled Environmental Films Show Geographic Variability and Host a Variety of Microorganisms

Environmental films are ubiquitous coatings that form on virtually all surfaces exposed to the troposphere. This work analyzes large molecular weight species in native environmental films acquired at three collection sites in the vicinity of Iowa City, Iowa, USA that include an urban residence, a wooded area, and a metropolitan building rooftop. The films are developed for up to twelve months on polished silicon wafer substrates housed in flat-plate passive samplers. Film composition was assessed by Fourier transform ion cyclotron resonance secondary ion mass spectrometry (FTICR-SIMS), and spatially resolved chemical morphology was analyzed via timeof- flight secondary ion mass spectrometry (ToF-SIMS) imaging. Mass analysis spanning up to m/z 2,000 included detection of a variety of organic and inorganic compounds. We characterize MS data with PCA to identify variability between the collection sites and the respective films generated therein, where we detected a variety of organic and biotically generated species consistent with fungal and bacterial microorganisms. Here, the film collection site as well as deposition time contribute to spectral variability, suggesting that geographic location as well as seasonal diversity may affect composition. SIMS analyses were complemented with microscopy and SYBR-Safe staining to highlight biological microorganisms within the films, which include evidence of fungi, protozoa, and bacteria. SIMS imaging also reveals domains concentrated with fragments indicative of proteinaceous macromolecules and microbial biomass, characteristic of biological presence within the film.

42 ENGINEERING↗

Community dynamics and functional characteristics of naphthalene‐degrading populations in contaminated surface sediments and hypoxic/anoxic groundwater

Summary Earlier research on the biogeochemical factors affecting natural attenuation in coal‐tar contaminated groundwater, at South Glens Falls, NY, revealed the importance of anaerobic metabolism and trophic interactions between degrader and bacterivore populations. Field‐based characterizations of both phenomena have proven challenging, but advances in stable isotope probing (SIP), single‐cell imaging and shotgun metagenomics now provide cultivation‐independent tools for their study. We tracked carbon from 13 C‐labelled naphthalene through microbial populations in contaminated surface sediments over 6 days using respiration assays, secondary ion mass spectrometry imaging and shotgun metagenomics to disentangle the contaminant‐based trophic web. Contaminant‐exposed communities in hypoxic/anoxic groundwater were contrasted with those from oxic surface sediments to identify putative features of anaerobic catabolism of naphthalene. In total, six bacteria were responsible for naphthalene degradation. Cupriavidus , Ralstonia and Sphingomonas predominated at the earliest stages of SIP incubations and were succeeded in later stages by Stenotrophomonas and Rhodococcus. Metagenome‐assembled genomes provided evidence for the ecological and functional characteristics underlying these temporal shifts. Identical species of Stenotrophomonas and Rhodococcus were abundant in the most contaminated, anoxic groundwater. Apparent increases in bacterivorous protozoa were observed following exposure to naphthalene, though insignificant amounts of carbon were transferred between bacterial degraders and populations of secondary feeders.

Wilhelm, Roland C.↗

Axiom Microbiome Array, the next generation microarray for high-throughput pathogen and microbiome analysis

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been utilized to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. In this study, the Axiom Microbiome Array was evaluated to discover its sensitivity, specificity and utility in microbiome analysis of veterinary clinical samples. The array contains probes designed to detect more than 12,000 species of viruses, bacteria, fungi, protozoa and archaea, yielding the most comprehensive microbial detection platform built to date. The array was able to detect Shigella and Aspergillus at 100 genome copies, and vaccinia virus DNA at 1,000 genome copies. The Axiom Microbiome Array made correct species-level calls in mock microbial community samples. When tested against serum, tissue, and fecal samples from pigs experimentally co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2, the microarray correctly detected these two viruses and other common viral and bacterial microbiome species. This cost-effective and high-throughput microarray is an efficient tool to rapidly analyze large numbers of clinical and environmental samples for the presence of multiple viral and bacterial pathogens.

59 BASIC BIOLOGICAL SCIENCES↗

Senescent changes in the ribosomes of animal cells in vivo and in vitro

The paper examines RNA-ribosomal changes observed in protozoa and fixed postmitotic cells, as well as the characteristics of intermitotic cells. Attention is given to a discussion of the implications of the reported ribosomal changes as to the senescent deterioration of protein synthesis and physiological functions. A survey of the literature suggests that, while the data on ribosomal change in dividing cells both in vivo and in vitro are inconclusive, there is strong histological and biochemical evidence in favor of some degree of quantitative ribosomal loss in fixed postmitotic cells. Since these decreases in ribosomes are demonstrated in differential cells from nematodes, insects and mammals, they may represent a universal manifestation of cytoplasmic senescence in certain types of fixed postmitotic animal cells. The observed variability in ribosomal loss for cells belonging to the same type suggests that this involution phenomenon is rather related to the wear and tear suffered by a particular cell.

Miquel, J.↗

Space Station and the life sciences

Previous fundamental research in space life sciences is examined, and consideration is devoted to studies relevant to Space Station activities. Microgravity causes weight loss, hemoconcentration, and orthostatic intolerance when astronauts returns to earth. Losses in bone density, bone calcium, and muscle nitrogen have also been observed, together with cardiovascular deconditioning, fluid-electrolyte metabolism alteration, and space sickness. Experiments have been performed with plants, bacteria, fungi, protozoa, tissue cultures, invertebrate species, and with nonhuman vertebrates, showing little effect on simple cell functions. The Spacelab first flight will feature seven life science experiments and the second flight, two. Further studies will be performed on later flights. Continued life science studies to optimize human performance in space are necessary for the efficient operation of a Space Station and the assembly of large space structures, particularly in interaction with automated machinery.

White, R. J.↗

A gravitational test of wave reinforcement versus fluid density models

Spermatozoa, protozoa, and algae form macroscopic patterns somewhat analogous to thermally driven convection cells. These bioconvective patterns have attracted interest in the fluid dynamics community, but whether in all cases these waves were gravity driven was unknown. There are two conflicting theories, one gravity dependent (fluid density model), the other gravity independent (wave reinforcement theory). The primary objectives of the summer faculty fellows were to: (1) assist in sample collection (spermatozoa) and preparation for the KC-135 research airplane experiment; and (2) to collaborate on ground testing of bioconvective variables such as motility, concentration, morphology, etc., in relation to their macroscopic patterns. Results are very briefly given.

Johnson, Jacqueline Umstead↗

Gravity effects on reproduction, development, and aging

The effects of various levels of gravity force (obtained by rotation in clinostats or by centrifugation) and the near-weightlessness condition aboard orbiting spacecraft on the fertilization, embryonic development, maturation, and aging of animals are examined. Results obtained from the American and Soviet spaceborne biology experiments are presented including those on mammals, amphibians, fish, birds, invertebrates, and protozoa. Theoretical issues related to the effect of gravity on various physiological systems are discused together with the future research goals concerning human life in space. It is noted that life in space (after adaptation to near-weightlessness) might be significantly prolonged due to a reduction in metabolic rate and a concomitant decrease in oxygen radical reactions.

Miquel, Jaime↗