Critical assessment of DNA adenine methylation in eukaryotes using quantitative deconvolution.

The discovery of N6-methyldeoxyadenine (6mA) across eukaryotes led to a search for additional epigenetic mechanisms. However, some studies have highlighted confounding factors that challenge the prevalence of 6mA in eukaryotes. We developed a metagenomic method to quantitatively deconvolve 6mA events from a genomic DNA sample into species of interest, genomic regions, and sources of contamination. Applying this method, we observed high-resolution 6mA deposition in two protozoa. We found that commensal or soil bacteria explained the vast majority of 6mA in insect and plant samples. We found no evidence of high abundance of 6mA in Drosophila, Arabidopsis, or humans. Plasmids used for genetic manipulation, even those from Dam methyltransferase mutant Escherichia coli, could carry abundant 6mA, confounding the evaluation of candidate 6mA methyltransferases and demethylases. On the basis of this work, we advocate for a reassessment of 6mA in eukaryotes.

Chromatin as a key consumer in the metabolite economy.

In eukaryotes, chromatin remodeling and post-translational modifications (PTMs) shape the local chromatin landscape to establish permissive and repressive regions within the genome, orchestrating transcription, replication, and DNA repair in concert with other epigenetic mechanisms. Though cellular nutrient signaling encompasses a huge number of pathways, recent attention has turned to the hypothesis that the metabolic state of the cell is communicated to the genome through the type and concentration of metabolites in the nucleus that are cofactors for chromatin-modifying enzymes. Importantly, both epigenetic and metabolic dysregulation are hallmarks of a range of diseases, and this metabolism-chromatin axis may yield a well of new therapeutic targets. In this Perspective, we highlight emerging themes in the inter-regulation of the genome and metabolism via chromatin, including nonenzymatic histone modifications arising from chemically reactive metabolites, the expansion of PTM diversity from cofactor-promiscuous chromatin-modifying enzymes, and evidence for the existence and importance of subnucleocytoplasmic metabolite pools.

Assessment of prokaryote to eukaryote ratios in environmental samples by SSU rDNA length polymorphism.

Microbial communities are important regulators of many processes in all ecosystems. Understanding of ecosystem processes requires at least an overview of the involved microorganisms. While in-depth identification of microbial species in environmental samples can be achieved by next generation sequencing, profiling of whole microbial communities can be accomplished via less labour-intensive approaches. Especially automated ribosomal intergenic spacer analysis (ARISA) are of interest as they are highly specific even at fine scales and widely applicable for environmental samples. Yet, established protocols lack the possibility to compare prokaryotic and eukaryotic communities as different primer sets are necessary. However, shifts in the eukaryote to prokaryote ratio can be a useful indicator for ecosystem processes like decomposition or nutrient cycling. We propose a protocol to analyse prokaryotic and eukaryotic communities using a single primer pair based reaction based on a region with variable length (V4, which is about 180 bp shorter in prokaryotes compared to eukaryotes) in the small ribosomal subunit flanked by two highly conservative regions. Shifts in the prokaryotic and eukaryotic ratio between samples can be reliably detected by fragment length polymorphism analysis as well as sequencing of this region. Together with established approaches such as ARISA or 16S and ITS rDNA sequencing, this can provide a more complex insight into microbial community shifts and ecosystem processes.

IT'S 2 for the price of 1: Multifaceted ITS2 processing machines in RNA and DNA maintenance.

Cells utilize sophisticated RNA processing machines to ensure the quality of RNA. Many RNA processing machines have been further implicated in regulating the DNA damage response signifying a strong link between RNA processing and genome maintenance. One of the most intricate and highly regulated RNA processing pathways is the processing of the precursor ribosomal RNA (pre-rRNA), which is paramount for the production of ribosomes. Removal of the Internal Transcribed Spacer 2 (ITS2), located between the 5.8S and 25S rRNA, is one of the most complex steps of ribosome assembly. Processing of the ITS2 is initiated by the newly discovered endoribonuclease Las1, which cleaves at the C2 site within the ITS2, generating products that are further processed by the polynucleotide kinase Grc3, the 5'→3' exonuclease Rat1, and the 3'→5' RNA exosome complex. In addition to their defined roles in ITS2 processing, these critical cellular machines participate in other stages of ribosome assembly, turnover of numerous cellular RNAs, and genome maintenance. Here we summarize recent work defining the molecular mechanisms of ITS2 processing by these essential RNA processing machines and highlight their emerging roles in transcription termination, heterochromatin function, telomere maintenance, and DNA repair.

Classification and Lineage Tracing of SH2 Domains Throughout Eukaryotes.

Today there exists a rapidly expanding number of sequenced genomes. Cataloging protein interaction domains such as the Src Homology 2 (SH2) domain across these various genomes can be accomplished with ease due to existing algorithms and predictions models. An evolutionary analysis of SH2 domains provides a step towards understanding how SH2 proteins integrated with existing signaling networks to position phosphotyrosine signaling as a crucial driver of robust cellular communication networks in metazoans. However organizing and tracing SH2 domain across organisms and understanding their evolutionary trajectory remains a challenge. This chapter describes several methodologies towards analyzing the evolutionary trajectory of SH2 domains including a global SH2 domain classification system, which facilitates annotation of new SH2 sequences essential for tracing the lineage of SH2 domains throughout eukaryote evolution. This classification utilizes a combination of sequence homology, protein domain architecture and the boundary positions between introns and exons within the SH2 domain or genes encoding these domains. Discrete SH2 families can then be traced across various genomes to provide insight into its origins. Furthermore, additional methods for examining potential mechanisms for divergence of SH2 domains from structural changes to alterations in the protein domain content and genome duplication will be discussed. Therefore a better understanding of SH2 domain evolution may enhance our insight into the emergence of phosphotyrosine signaling and the expansion of protein interaction domains.

Evolution of condensin and cohesin complexes driven by replacement of Kite by Hawk proteins.

Mitotic chromosome condensation, sister chromatid cohesion, and higher order folding of interphase chromatin are mediated by condensin and cohesin, eukaryotic members of the SMC (structural maintenance of chromosomes)-kleisin protein family. Other members facilitate chromosome segregation in bacteria [1]. A hallmark of these complexes is the binding of the two ends of a kleisin subunit to the apices of V-shaped Smc dimers, creating a tripartite ring capable of entrapping DNA (Figure 1A). In addition to creating rings, kleisins recruit regulatory subunits. One family of regulators, namely Kite dimers (Kleisin interacting winged-helix tandem elements), interact with Smc-kleisin rings from bacteria, archaea and the eukaryotic Smc5-6 complex, but not with either condensin or cohesin [2]. These instead possess proteins containing HEAT (Huntingtin/EF3/PP2A/Tor1) repeat domains whose origin and distribution have not yet been characterized. Using a combination of profile Hidden Markov Model (HMM)-based homology searches, network analysis and structural alignments, we identify a common origin for these regulators, for which we propose the name Hawks, i.e. HEAT proteins associated with kleisins.

Further assessment of the protozoal contribution to the nutrition of the ruminant animal.

The flow of protozoa from the reticulo-rumen is lower than expected, due to ability of protozoa to prevent washout through sequestration on feed particles and the rumen epithelium. In order to estimate the distribution of protozoa within the reticulo-rumen and passage to the omasum, Czerkawski (1987) developed a model containing pools for the rumen liquid phase, rumen solid phase, and the omasum. This model was used to estimate loss of protozoa in the omasum as well as the amount of protozoal protein available to the animal in the lower gut. A number of assumptions were incorporated into the model, some of which appear unsupported by current research. This paper represents an update, revision, and re-evaluation of Czerkawski's model, where the assumptions that all protozoa in the 'attached' phase are in solid digesta, and that protozoa only leave the rumen in the liquid, have been relaxed. Therefore, the revised model allows for sequestration of protozoa on the rumen epithelium and protozoal passage with particulate outflow. Using experimental observations with inputs within biological limits, the revised model and Czerkawski's original model were verified. The effect of diet on the model was then assessed using inputs from a 100% forage diet and a 35-65% concentrate diet. The extent of sequestration was also varied from complete sequestration, to partial sequestration, and no sequestration. A sensitivity analysis was conducted through a linear regression of perturbed mean inputs versus outputs. The results from the revised model indicate that within the reticulo-rumen, the concentrate diet has a greater fractional flow rate of protozoa from the liquid to solid phase, but a lesser fractional flow rate back to the liquid phase, compared to the forage diet. As well, the concentrate diet has a slower net growth rate of protozoa in the attached phase, compared to the forage diet. In the omasum, the forage diet has a less negative net growth rate, compared to the concentrate diet. The forage diet was also associated with smaller loss of protozoa from the omasum. There are limited data from the omasum to be incorporated into the revised model, especially for quantity of protozoa in the omasum. Further research on quantification of protozoa in the omasum could strengthen the predictions made by the model. Despite this, the revised model found a loss of protozoa in the omasum similar to that suggested by Czerkawski's original model 65-73% versus 66%. The revised model results indicate that efforts to increase protozoal flow to the duodenum should focus on reduced sequestration and increased outflow rate from the rumen, although more research is needed to quantify protozoa in the omasum, and to investigate the role of sequestration onto the wall of the reticulo-rumen.

The Role of Heterotrophic Microbial Communities in Estuarine C Budgets and the Biogeochemical C Cycle with Implications for Global Warming: Research Opportunities and Challenges.

Estuaries are among the most productive and economically important marine ecosystems at the land-ocean interface and contribute significantly to exchange of CO2 with the atmosphere. Estuarine microbial communities are major links in the biogeochemical C cycle and flow of C in food webs from primary producers to higher consumers. Considerable attention has been given to bacteria and autotrophic eukaryotes in estuarine ecosystems, but less research has been devoted to the role of heterotrophic eukaryotic microbes. Current research is reviewed here on the role of heterotrophic eukaryotic microbes in C biogeochemistry and ecology of estuaries, with particular attention to C budgets, trophodynamics, and the metabolic fate of C in microbial communities. Some attention is given to the importance of these processes in climate change and global warming, especially in relation to sources and sinks of atmospheric CO2 , while also documenting the current paucity of research on the role of eukaryotic microbes that contribute to this larger question of C biogeochemistry and the environment. Some recommendations are made for future directions of research and opportunities of applying newer technologies and analytical approaches to a more refined analysis of the role of C in estuarine microbial community processes and the biogeochemical C cycle.

Wastewater treatment high rate algal ponds (WWT HRAP) for low-cost biofuel production.

Growing energy demand and water consumption have increased concerns about energy security and efficient wastewater treatment and reuse. Wastewater treatment high rate algal ponds (WWT HRAPs) are a promising technology that could help solve these challenges concurrently where climate is favorable. WWT HRAPs have great potential for biofuel production as a by-product of WWT, since the costs of algal cultivation and harvest for biofuel production are covered by the wastewater treatment function. Generally, 800-1400 GJ/ha/year energy (average biomass energy content: 20 GJ/ton; HRAP biomass productivity: 40-70 tons/ha/year) can be produced in the form of harvestable biomass from WWT HRAP which can be used to provide community-level energy supply. In this paper the benefits of WWT HRAPs are compared with conventional mass algal culture systems. Moreover, parameters to effectively increase algal energy content and overall energy production from WWT HRAP are discussed including selection of appropriate algal biomass biofuel conversion pathways.

Nutrient removal from agricultural drainage water using algal turf scrubbers and solar power.

The objectives of this study were to determine nutrient removal rates and costs using solar-powered algal turf scrubber (ATS) raceways and water from an agricultural drainage ditch. Algal productivity using daytime-only flow was 3-lower compared to productivity using continuous flow. Results from this and other studies suggest a non-linear relationship between flow rate and nitrogen removal rates. Nitrogen (N) and phosphorus (P) removal rates averaged 125 mg N, 25 mg P m(-2) d(-1) at the highest flow rates. Nutrient removal rates were equivalent to 310 kg N and 33 kg P ha(-1) over a 7 month season. Projected nutrient removal costs ($90-$110 kg(-1) N or $830-$1050 kg(-1) P) are >10-fold higher than previous estimates for ATS units used to treat manure effluents.

Establishment of microbial eukaryotic enrichment cultures from a chemically stratified antarctic lake and assessment of carbon fixation potential.

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter (1). These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms (2). Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling (3) and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms (4, 2). Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web (5). Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism (6, 7). Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited (8, 4, 9, 10, 5). A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle. We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity (4, 11), as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms. RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs (12). In this study, we applied a radioisotope assay modified for filtered samples (13) to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins.

Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites in proteins is currently lacking. We have therefore analyzed the occurrence and occupancy of phosphorylated sites (~100,281) in a large set of eukaryotic proteins (~22,995). Phosphorylation probability was found to be much higher in both the termini of protein sequences and this is much pronounced in transmembrane proteins. A large proportion (51.3%) of occupied sites had a nearby phosphorylation within a distance of 10 amino acids; however, this proportion is very high compared to the expected one (16.9%). The distribution of phosphorylated sites in proteins showed a strong deviation from the expected maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins.

Evalúan las probables propiedades terapéuticas de la espirulina / Assessment of the probable therapeutic properties of spirulina

La espirulina es un alga verdeazulada (cianobacteria) que ha sido consumida por los seres humanos durante cientos de años en la región Kanem de Chad y en las regiones lacustres de México. Actualmente se comercializa en todo el mundo como alimento terapéutico. Su potencial como tratamiento de varias enfermedades se encuentra en evaluación. El consumo mundial de espirulina ha clarificado tanto sus potenciales efectos adversos como sus acciones beneficiosas. En este artículo se presenta un breve resumen del uso de la espirulina en el área de la salud.

Combinatorial life cycle assessment to inform process design of industrial production of algal biodiesel.

The use of algae as a feedstock for biodiesel production is a rapidly growing industry, in the United States and globally. A life cycle assessment (LCA) is presented that compares various methods, either proposed or under development, for algal biodiesel to inform the most promising pathways for sustainable full-scale production. For this analysis, the system is divided into five distinct process steps: (1) microalgae cultivation, (2) harvesting and/or dewatering, (3) lipid extraction, (4) conversion (transesterification) into biodiesel, and (5) byproduct management. A number of technology options are considered for each process step and various technology combinations are assessed for their life cycle environmental impacts. The optimal option for each process step is selected yielding a best case scenario, comprised of a flat panel enclosed photobioreactor and direct transesterification of algal cells with supercritical methanol. For a functional unit of 10 GJ biodiesel, the best case production system yields a cumulative energy demand savings of more than 65 GJ, reduces water consumption by 585 m(3) and decreases greenhouse gas emissions by 86% compared to a base case scenario typical of early industrial practices, highlighting the importance of technological innovation in algae processing and providing guidance on promising production pathways.

Algal biodiesel economy and competition among bio-fuels.

This investigation examines the possible results of policy support in developed and developing economies for developing algal biodiesel through to 2040. This investigation adopts the Taiwan General Equilibrium Model-Energy for Bio-fuels (TAIGEM-EB) to predict competition among the development of algal biodiesel, bioethanol and conventional crop-based biodiesel. Analytical results show that algal biodiesel will not be the major energy source in 2040 without strong support in developed economies. In contrast, bioethanol enjoys a development advantage relative to both forms of biodiesel. Finally, algal biodiesel will almost completely replace conventional biodiesel. CO(2) reduction benefits the development of the bio-fuels industry.

The role of biochemical engineering in the production of biofuels from microalgae.

Environmental changes that have occurred due to the use of fossil fuels have driven the search for alternative sources that have a lower environmental impact. First-generation biofuels were derived from crops such as sugar cane, corn and soybean, which contribute to water scarcity and deforestation. Second-generation biofuels originated from lignocellulose agriculture and forest residues, however these needed large areas of land that could be used for food production. Based on technology projections, the third generation of biofuels will be derived from microalgae. Microalgae are considered to be an alternative energy source without the drawbacks of the first- and second-generation biofuels. Depending upon the growing conditions, microalgae can produce biocompounds that are easily converted into biofuels. The biofuels from microalgae are an alternative that can keep the development of human activity in harmony with the environment. This study aimed to present the main biofuels that can be derived from microalgae.

Economy of operon formation: cotranscription minimizes shortfall in protein complexes.

Genes of prokaryotes and Archaea are often organized in cotranscribed groups, or operons. In contrast, eukaryotic genes are generally transcribed independently. Here we show that there is a substantial economic gain for the cell to cotranscribe genes encoding protein complexes because it synchronizes the fluctuations, or noise, in the levels of the different components. This correlation substantially reduces the shortfall in production of the complex. This benefit is relatively large in small cells such as bacterial cells, in which there are few mRNAs and proteins per cell, and is diminished in larger cells such as eukaryotic cells.

An outlook on microalgal biofuels.

Microalgae are considered one of the most promising feedstocks for biofuels. The productivity of these photosynthetic microorganisms in converting carbon dioxide into carbon-rich lipids, only a step or two away from biodiesel, greatly exceeds that of agricultural oleaginous crops, without competing for arable land. Worldwide, research and demonstration programs are being carried out to develop the technology needed to expand algal lipid production from a craft to a major industrial process. Although microalgae are not yet produced at large scale for bulk applications, recent advances-particularly in the methods of systems biology, genetic engineering, and biorefining-present opportunities to develop this process in a sustainable and economical way within the next 10 to 15 years.

A cost/effective screening method for assessing the toxicity of nutrient rich effluents to algae.

Screening whole effluent toxicity tests are cost/effective methods for detecting the presence of toxic concentrations of unknown pollutants, but the application must solve the problem associated with the effect of high and variable concentrations of nutrients in the effluent on the results of algal toxicity tests. This work proposes a cost/effective test, based on three dilution levels measured at a single point time and a discriminant model for establishing if this kind of complex samples, with difficult interpretation of dilution-response curves, should be considered toxic to algae. This procedure identified properly around 85% of the samples considered toxic by expert judgement.

Genome streamlining and the elemental costs of growth.

Pervasive relationships between growth rate, genome size and RNA content exist. One interesting potential consequence of these interrelationships is that selection for high growth rate should be associated with small genomes and high RNA content. Here, we use phosphorus (P) and nitrogen (N) demands of growth along with nucleic acid production as the currency to explore the interrelationships between growth rate and genome size in eukaryotes. We argue that reallocation of P (and eventually N) from DNA to RNA under sustained selection for rapid growth in nutritionally limited environments can lead to genome streamlining in eukaryotes, and that this mechanism might contribute to the evolution of reduced genome size.